Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor

ABSTRACT

Methods and compositions are disclosed that are useful for the prevention and/or treatment of cardiovascular and cardiac diseases and disorders, or damage resulting from surgical or medical procedures that may cause ischemic or ischemic/reperfusion damage in humans; and cardiovascular trauma. The beneficial effects of the compositions and methods are achieved through the use of pharmaceutical compositions that include agents that interfere with the production and/or biological activities of sphingolipids and their metabolites, particularly sphingosine (SPH) and sphingosine-1-phosphate (S-1-P). Also disclosed are methods for identifying and isolating therapeutic agents.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. patent application Ser.No. 60/257,926 entitled “Compositions and Methods for the Treatment andPrevention of Cardiac and Myocardial Disorders” by Sabbadini, Roger A.,filed Dec. 22, 2000.

[0002] This application is related to U.S. patent application Ser. No.______ (attorney docket No. 078853-0302), Ser. No. ______ (attorneydocket No. 078853-0304), and Ser. No. ______ (attorney docket No.078853-0305), each entitled “Compositions and Methods for the Treatmentand Prevention of Cardiovascular Diseases and Disorders, and forIdentifying Agents Therapeutic Therefor” by Sabbadini, Roger A., andfiled Dec. 21, 2001.

[0003] All of the preceding applications are hereby incorporated intheir entirety by reference thereto.

FIELD OF THE INVENTION

[0004] The invention relates generally to the area of treatment and/orprevention of cardiovascular and cerebrovascular diseases, disorders and physical trauma. The beneficial effect of the invention is achievedthrough the use of pharmaceutical compositions that contain agents thatinterfere with the production and/or biological activities ofsphingolipids and their metabolites. The invention is also drawn tomethods for isolating, formulating and using pharmaceuticalcompositions, and kits and medical devices comprising such compositions.

BACKGROUND OF THE INVENTION

[0005] The following description includes information that may be usefulin understanding the present invention. It is not an admission that anyof the information provided herein, or any publication specifically orimplicitly referenced herein, is prior art, or even particularlyrelevant, to the presently claimed invention.

[0006] Cardiovascular Diseases and Disorders

[0007] Ischemic heart disease is the leading cause of death in the U.S.Each year approximately 1.5 million people suffer heart attacks(myocardial infarctions), of which ⅓ (i.e. about 500,000) are fatal. Inaddition, about 6.75 million Americans suffer from angina pectoris, themost common manifestation of cardiac ischemia. Angina pectoris is apainful feeling of pressure in the chest that results from ischemicheart disease. In total, there are 13.5 million patients living withischemic heart disease in the U.S. Americans in the high-risk categoriesfor this disease include persons having one or more indicators/riskfactors therefor, including but not limited to hypertension, high levelsof serum cholesterol and a family history of heart disease. Many peoplehave at least one of these indicator/risk factors; for example, thereare 50 million Americans diagnosed with hypertension alone.

[0008] “Ischemia” is a condition associated with an inadequate flow ofoxygenated blood to a part of the body, typically caused by theconstriction or blockage of the blood vessels supplying it. Ischemiaoccurs any time that blood flow to a tissue is reduced below a criticallevel. This reduction in blood flow can result from: (i) the blockage ofa vessel by an embolus (blood clot); (ii) the blockage of a vessel dueto atherosclerosis; (iii) the breakage of a blood vessel (a bleedingstroke); (iv) the blockage of a blood vessel due to acutevasoconstriction; (v) a myocardial infarction (when the heart stops, theflow of blood to organs is reduced and ischemia results); (vi) trauma;(vii) surgery, during which blood flow to a tissue or organ needs to bereduced or stopped to achieve the aims of surgery (e.g., angioplasty,heart and lung/heart transplants); (viii) exposure to certain agents,e.g., dobutamine or adenosine (Lagerqvist et al., Br. Heart J.68:282-285, 1992) or (ix) anti-neoplastic and other chemotherapeuticagents, such as doxorubicin, that are cardiotoxic.

[0009] Even if the flow rate (volume/time) of blood is adequate,ischemia may nonetheless occur due to hypoxia. “Hypoxia” refers toconditions in which the oxygen content of blood is insufficient tosatisfy normal cellular oxygen requirements. Hypoxic blood is, bydefinition, distinct from normoxic blood, i.e., blood in which theoxygen content is sufficient to satisfy normal cellular oxygenrequirements. Such conditions include but are not limited to forms ofheart failure that adversely affect cardiac pumping such ashypertension, arrhythmias, septic shock, trauma, cardiomyopathies andcongestive heart disease.

[0010] Myocardial ischemic disorders occur when cardiac blood flow isrestricted (ischemia) and/or when oxygen supply to the heart muscle iscompromised (hypoxia) such that the heart's demand for oxygen is not metby the supply. Coronary artery disease (CAD) arising fromarteriosclerosis, particularly atherosclerosis, is the most common causeof ischemia, and has symptoms such as stable or unstable anginapectoris. CAD can lead to acute myocardial infarctions (AMI) and suddencardiac death. The spectrum of ischemic conditions which result in heartfailure is referred to as Acute Coronary Syndrome (ACS). Reperfusioninjury is often a consequence of ischemia, in particular whenanti-coagulants, thrombolytic agents, or anti-anginal medications areused or when the cardiac vasculature is surgically opened by angioplastyor by coronary artery grafting.

[0011] Cardiotoxic agents are those materials which would cause a lossof cardiac function, including negative inotropy, arthyTHmias, heartfailure, and cell death (both apoptotic and necrotic).

[0012] Presently, treatments for acute myocardial infarction and othercardiac diseases include but are not limited to mechanical devices andassociated procedures therewith (e.g., coronary angioplasty; Grines etal., N. Engl. J. Med. 3298:673-679, 1993); thrombolytic agents such asstreptokinase, tPA, and derivatives thereof. Adjuvants to thesetherapies include beta-blockers, aspirin and heparin, and glycoprotein(GP) IIb/IIIa inhibitors (Antman et al., Circ. 99:2720-2732, 1999). GPIIb/IIIa inhibitors decrease platelet aggregation and thrombus formation(for a review, see Topol, Lancet 353:227-231, 1999). Examples includebut are not limited to monoclonal antibodies (e.g., abciximab), cyclicpeptides (e.g., eptifibatide), and nonpeptide peptidomimetics (e.g.,tirofibian, lamifiban, xemilofiban, sibrafiban, and lefradafibian).

[0013] Preventive treatments include but are not limited to those thatreduce a patient's cholesterol levels by, e.g., diet management andpharmacological intervention. Statins are one type of agent that havebeen used to reduce cholesterol levels. Statins are believed to act byinhibiting the activity of HMG-CoA reductase, which in turn increasesthe hepatic production of cholesterol receptors (Nickenig et al., Circ.100:2131-2134, 1999). The hepatic cholesterol receptors bind cholesteroland remove it from blood. Such agents include but are not limited tolovastatin, simvastatin, pravastatin, fluvastatin (Lennernas, Clin.Pharmackinet. 32:403-425, 1997). These and other statins slows theprogression of coronary artery disease, and may induce regression ofatherosclerotic lesions in patients. It is not known, however, whetherother reductases are inhibited by such agents, and what side effectsmight occur as a result.

[0014] Cerebrovascular Diseases and Disorders

[0015] Patients experiencing cerebral ischemia often suffer fromdisabilities ranging from transient neurological deficit to irreversibledamage (stroke) or death. Cerebral ischemia, i.e., reduction orcessation of blood flow to the central nervous system, can becharacterized as either global or focal.

[0016] Focal cerebral ischemia refers to cessation or reduction of bloodflow within the cerebral vasculature resulting from a partial orcomplete occlusion in the intracranial or extracranial cerebralarteries. Such occlusion typically results in stroke, a syndromecharacterized by the acute onset of a neurological deficit that persistsfor at least 24 hours, reflecting focal involvement of the centralnervous system and is the result of a disturbance of the cerebralcirculation. Other causes of focal cerebral ischemia include vasospasmdue to subarachnoid hemorrhage or iatrogenic intervention.

[0017] Global cerebral ischemia refers to reduction of blood flow withinthe cerebral vasculature resulting from systemic circulatory failure.The failure of the circulatory system to maintain adequate cellularperfusion leads to a in reduction of oxygen and nutrients to tissues.Thus, global cerebral ischemia results from severe depression of cardiacperformance. The most frequent cause is acute myocardial infarction withloss of substantial muscle mass. Pump failure can also result from acutemyocarditis or from depression of myocardial contractility followingcardiac arrest or prolonged cardiopulmonary bypass. Mechanicalabnormalities, such as severe valvular stenosis, massive aortic ormitral regurgitation, acutely acquired ventricular septal defects, canalso reduce cardiac output. Additional causes include cardiacarrhythmia, such as ventricular fibrillation, and any cardiac diseasedescribed herein. Further causes include interventional procedures, suchas carotid angioplasty, stenting or endarterectomy, which mightotherwise result in focal cerebral ischemia, and also cardiac procedureswhich may result in global cerebral ischemia, such as cardiaccatheterization, electrophysiologic studies, and angioplasty.

[0018] Those skilled in the art are easily able to identify patientshaving a stroke or at risk of having a stroke, cerebral ischemia, headtrauma, or epilepsy. For example, patients who are at risk of having astroke include, but are not limited to, patients having hypertension orundergoing major surgery.

[0019] Traditionally, emergent management of acute ischemic strokeconsists of mainly general supportive care, e.g. hydration, monitoringneurological status, blood pressure control, and/or anti-platelet oranti-coagulation therapy. Heparin has been administered to strokepatients with limited and inconsistent effectiveness. In somecircumstances, the ischemia resolves itself over a period of time due tothe fact that some thrombi get absorbed into the circulation, orfragment and travel distally over a period of a few days. In 1996, theFood and Drug Administration approved the use of tissue plasminogenactivator (t-PA) or Activase®, for treating acute stroke. However,treatment with systemic t-PA is associated with increased risk ofintracerebral hemorrhage and other hemorrhagic complications. Aside fromthe administration of thrombolytic agents and heparin, there are notherapeutic options currently on the market for patients suffering fromocclusion focal cerebral ischemia. Vasospasm may be partially responsiveto vasodilating agents. The newly developing field of neurovascularsurgery, which involves placing minimally invasive devices within thecarotid arteries to physically remove the offending lesion may provide atherapeutic option for these patients in the future, although this kindof manipulation may lead to vasospasm itself.

[0020] Documents

[0021] U.S. Pat. No. 6,210,976 B1 and published PCT patent applicationW098/57179 (PCT/US98/10486), both entitled “Methods for Early Detectionof Heart Disease”, hereby incorporated by reference, relate to the useof blood levels of certain sphingolipids for screening for earlyischemic events before symptoms are presented in persons with high riskfor heart disease, or in a triage setting for patients with acutecoronary syndrome.

[0022] PCT Application PCT/US01/12706, published as WO 01/80903,entitled “Detection and Treatment of Atherosclerosis Based on PlasmaSphingomyelin Concentration”, relates to enzymatic methods to measureplasma and tissue sphingomyelin concentrations, and that human plasmasphingomyelin levels are positively correlated with atherosclerosis andcoronary heart disease.

[0023] U.S. Pat. No. 5,929,039, entitled “Method for Treating CardiacDysfunction and Pharmaceutical Compositions Useful Therefor”, relates todisclose methods methods for the prophylaxis or treatment of cardiacarrhythmia using an agent capable of blocking or inhibiting the effector release of inositol(1,4,5)trisphosphate in cardiac tissue. The agentmay be an aminoglycoside, including gentamicin.

[0024] U.S. Pat. No. 5,677,288, entitled “Use of Aminoglycosides toProtect Against Excitotoxic Neuron Damage”, relates to the use of anaminoglycoside, which may be gentamicin, that suppresses the flow ofcalcium ions into neurons through N-type calcium channels. The methodrelates to reducing excitotoxic damage to neurons, which can occur as aresult of stroke, cerebral ischemia/hypoxia, or other events orconditions.

[0025] Published U.S. patent application Ser. No. 20010041688, entitled“Methods and Compositions for the Regulation of Vasoconstriction”,relates to modulation of sphingosine kinase and sphingosine-1-phosphatephosphatase activity and EDG receptor signaling for the treatment ofconditions relating to vasoconstriction and vasoconstriction, includingmigraine, stroke, subarachnoid hemorrhage and vasospasm.

[0026] Ancellin et al., “Extracelluar export of sphingosine kinase-1enzyme: Sphingosine 1 phosphate generation and the induction ofangiogenic vascular maturation”, JBC Papers in Press. Published on Dec.10, 2001 as manuscript M102841200 relates to events related toangiogenosis that are mediated by a sphingosine kinase.

SUMMARY OF THE INVENTION

[0027] The invention is drawn to compositions and methods for treatingor preventing cardiovascular, cardiac, myocardial and other diseases,disorders or physical trauma, and/or cerbrovascular diseases anddisorders, in which therapeutic agents are administered to a patientthat alters the activity or concentration of an undesirable, toxicand/or cardiotoxic sphingolipids, or metabolites thereof. Thetherapeutic methods and compositions of the invention are said to be“sphingolipid-based” in order to indicate that they act by changing theabsolute, relative and/or available concentration and/or activities ofcertain undesirable, toxic or cardiotoxic sphingolipids. The inventionis also drawn to chemical libraries and screening assays that are usedto identify novel sphingolipid-based therapeutics.

[0028] The compositions of the invention are used in methods ofsphingolipid-based cardiovascular and cardiac therapy. “Cardiac therapy”refers to the prevention and/or treatment of myocardial diseases,disorders or physical trauma. Conditions of particular interest includebut not limited to myocardial ischemia; acute myocardial infarction(AMI); coronary artery disease (CAD); acute coronary syndrome (ACS);cardiac cell and tissue damage that may occur during or as a consequenceof pericutaneous revascularization (coronary angioplasty) with orwithout stenting; coronary bypass grafting (CABG) or other surgical ormedical procedures or therapies that may cause ischemic orischemic/reperfusion damage in humans; and cardiovascular trauma.

[0029] “Cardiovascular therapy” encompasses cardiac therapy as well asthe prevention and/or treatment of other diseases associated with thecardiovascular system, such as heart disease. The term “heart disease”encompasses any type of disease, disorder, trauma or surgical treatmentthat involves the heart or myocardial tissue. Of particular interest areheart diseases that relate to hypoxia and/or ischemia of myocardialtissue and/or heart failure. One type of heart disease that can resultfrom ischemia is reperfusion injury,such as can occur whenanti-coagulants, thrombolytic agents, or anti-anginal medications areused in therapy, or when the cardiac vasculature is surgically opened byangioplasty or by coronary artery grafting. Another type of heartdisease to which the invention is directed is coronary artery disease(CAD), which can arise from arteriosclerosis, particularlyatherosclerosis, a common cause of ischemia. CAD has symptoms such asstable or unstable angina pectoris, and can lead to acute myocardialinfarctions (AMI) and sudden cardiac death. The term “heart failure”encompasses acute myocardial infarction, myocarditis, a cardiomyopathy,congestive heart failure, septic shock, cardiac trauma and idopathicheart failure. The spectrum of ischemic conditions which result in heartfailure is referred to as Acute Coronary Syndrome (ACS).

[0030] “Cerebrovascular therapy” refers to therapy directed to theprevention and/or treatment of diseases and disorders associated withcerebral ischemia and/or hypoxia. Of particular interest is cerebralischemia and/or hypoxia resulting from global ischemia resulting from aheart disease, including without limitation heart failure.

[0031] “Toxic sphingolipids” are those sphingolipids that can cause orenhance the necrosis and/or apoptosis of cells, including, in someinstances, particular cell types that are found in specific tissues ororgans. “Cardiotoxic sphingolipids” are toxic sphingolipids thatdirectly or indirectly cause or enhance cardiac arrythmias, the negativeinotropy (loss of contractile function) of the heart and the necrosisand/or apoptosis of cells found in or associated with the heart,including but not limited to cardiomyocytes, cardiac neurons and thelike. “Undesirable sphingolipids” include toxic and cardiotoxicsphingolipids, as well as metabolites, particularly metabolicprecursors, of toxic and cardiotoxic sphingolipids. Undesirable,cardiotoxic and/or toxic sphingolipids of particular interest includebut are not limited to ceramide (CER), sphingosine-1-phosphate (S-1-P)and sphingosine (SPH; D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol,or sphinganine).

[0032] The term “metabolites” refers to compounds from whichsphingolipids are made, as well as those that result from thedegradation of sphingolipids; that is compounds that are involved in thesphingolipid metabolic pathways (FIGS. 1 and 2). Metabolites includemetabolic precursors and metabolic products. The term “metabolicprecursors” refers to compounds from which sphingolipids are made.Metabolic precursors of particular interest include but are not limitedto SPC, sphingomyelin, dihydrosphingosine, dihydroceramide, and3-ketosphiganine. The term “metabolic products” refers to compounds thatresult from the degradation of sphingolipids, such as phosphorylcholine(a.k.a. phosphocholine, choline phosphate), fatty acids, including freefatty acids, and hexadecanal (a.k.a. palmitaldehyde).

[0033] As used herein, the term “therapeutic” encompasses the fallspectrum of treatments for a disease or disorder. A “therapeutic” agentof the invention may act in a manner that is prophylactic or preventive,including those that incorporate procedures designed to targetindividuals that can be identified as being at risk (pharmacogenetics);or in a manner that is ameliorative or curative in nature; or may act toslow the rate or extent of the progression of a disease or disorder; ormay act to minimize the time required, the occurrence or extent of anydiscomfort or pain, or physical limitations associated with recuperationfrom a disease, disorder or physical trauma; or may be used as anadjuvant to other therapies and treatments. The term “cardiotherapeuticagent” refers to an agent that is therapeutic to diseases and diseasescaused by or associated with cardiac and mycardial diseases anddisorders.

[0034] Without wishing to be bound by any particular theory, it isbelieved that the level of undesirable sphingolipids such as SPH orS-1-P, and/or one or more of their metabolites, cause or contribute tothe development of cardiac and myocardial diseases and disorders.Because sphingolipids are also involved in fibrogenesis and woundhealing of liver tissue (Davaille et al., J. Biol. Chem.275:34268-34633, 2000; Ikeda et al., Am J. Physiol. Gastrointest. LiverPhysiol 279:G304-G310, 2000), healing of wounded vasculatures (Lee etal., Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000), and otherdisease states or disorders, or events associated with such diseases ordisorders, such as cancer, angiogenesis and inflammation (Pyne et al.,Biochem. J. 349:385-402, 2000), the compositions and methods of thepresent disclosure may be applied to treat these diseases and disordersas well as cardiac and myocardial diseases and disorders.

[0035] One form of sphingolipid-based therapy involves manipulating themetabolic pathways of sphingolipids in order to decrease the actual,relative and/or available in vivo concentrations of undesirable, toxicand/or cardiotoxic sphingolipids. The invention provides compositionsand methods for treating or preventing cardiac and myocardial diseases,disorders or physical trauma, in which therapeutic agents areadministered to a patient that alters the activity or concentration ofan enzyme, wherein the enzyme catalyzes a reaction that produces ordegrades undesirable, toxic and/or cardiotoxic sphingolipids, ormetabolites thereof. An “enzyme” is a protein or polypeptide thatcatalyzes (causes, accelerates or enhances) a chemical reaction. Theterm “metabolism” is used to describe the biological construction ordestruction of a compound. Metabolism comprises the synthesis(constructive metabolism, a.k.a. anabolism) of compounds and thedegradation (destructive metabolism, a.k.a. catabolism) thereof. Enzymesof particular interest, and preferred modulating agents thereof(inhibitors/activators or stimulators/blocking agents), are described inthe Detailed Description (see also Examples 7 through 10).

[0036] In one version of this form of sphingolipid-based therapy,metabolic steps that involve the production of sphingolipids areinhibited or blocked. Therapeutic agents and methods are used todecrease the amount or activity of enzymes that catalyze chemicalreactions that degrade undesirable sphingolipids and/or metabolicprecursors thereof. Thus, net sphingolipid catabolism is increased.

[0037] In another version of this form of sphingolipid-based therapy,metabolic steps that involve the destruction of sphingolipids areactivated or stimulated. Therapeutic agents and methods are used toincrease the amount or activity of enzymes that catalyze chemicalreactions that degrade undesirable sphingolipids and/or metabolicprecursors thereof. Thus, net sphingolipid anabolism is decreased.

[0038] One form of sphingolipid-based therapy involves the use of agentsthat bind undesirable, toxic and/or cardiotoxic sphingolipids, ormetabolites thereof. Such sphingolipid-binding agents include but arenot limited to proteins and polypeptide derivatives thereof that bindundesirable, toxic and/or cardiotoxic sphingolipids or metabolitesthereof. Such a protein and polypeptide may, by way of non-limitingexample, be a non-catalytic derivative of an enzyme involved in thesphingolipid metabolic pathways, a derivative of proteins thatparticipate in the sphingomyelin signaling pathway, a derivative of areceptor that binds an undesirable, toxic and/or cardiotoxicsphingolipid, an antibody or antibody derivative that is directed to(specifically binds) an undesirable, toxic and/or cardiotoxicsphingolipid. Such derivatives are preferably water soluble.(Sphingolipid-binding agents are described in the Detailed Descriptionof the Invention; see also Examples 6 and 14).

[0039] One form of sphingolipid-based therapy involves the use of agentsthat bind sphingolipid receptors that initiate and stimulate thesphingomyelin signaling pathway. This pathway ultimately results inincreased ceramide production. An increased level of ceramide would, inturn, be expected to result in elevated concentrations of undesirablesphingolipids such as, e.g., S-1-P and SPH. Thus, inhibiting or blockingsuch receptors decreases, or at least prevents an increase due to thesphingomyelin signaling pathway, the intracellular production ofceramide and metabolites thereof (see the Detailed Description andExample 9). Another form of sphingolipid-based therapy involves the useof molecular genetics to generate therapeutic agents (see the DetailedDescription and Example 18).

[0040] In one version of this form of sphingolipid-based therapy, thetherapeutic agent is a protein (including, without limitation,polypeptides, oligopeptides, and peptidomimetics). A “protein” is amolecule having a sequence of amino acids that are linked to each otherin a linear molecule by peptide bonds. The term protein refers to apolypeptide that is isolated from a natural source, or produced from anisolated cDNA using recombinant DNA technology; and has a sequence ofamino acids having a length of at least about 200 amino acids. As usedherein, the term “polypeptide” includes proteins, fusion proteins,oligopeptides and polypeptide derivatives, with the exception thatpeptidomimetics are considered to be small molecules herein. An“oligopeptide” is a polypeptide having a short amino acid sequence(i.e., 2 to about 200 amino acids). An oligopeptide is generallyprepared by chemical synthesis. Although oligopeptides and proteinfragments may be otherwise prepared, it is possible to use recombinantDNA technology and/or in vitro biochemical manipulations. For example, anucleic acid encoding an amino acid sequence may be prepared and used asa template for in vitro transcription/translation reactions.

[0041] A “protein fragment” is a proteolytic fragment of a largerpolypeptide, which may be a protein or a fusion protein. A proteolyticfragment may be prepared by in vivo or in vitro proteolytic cleavage ofa larger polypeptide, and is generally too large to be prepared bychemical synthesis. Preferably, proteolytic fragments have amino acidsequences having a length from about 10 to about 5,000 amino acids; morepreferably about 200 to 1000 amino acids; most preferably 200 to about1,000 amino acids.

[0042] A therapeutic protein may be a dominant negative mutant of anenzyme that catalyzes a reaction that results in the production of anundesirable, toxic and/or cardiotoxic sphingolipid or a metabolitethereof, of a receptor for such a sphingolipid, or of a protein thatparticipates in the sphingomyelin signaling pathway. A “dominantnegative mutant protein” is one that, when expressed, (1) does notitself provide the activity of the wildtype protein and (ii) inhibitsthe action of the wildtype form of the protein. The therapeutic proteinmay be an enzyme, produced by recombinant DNA technology or any otherappropriate method, that catalyzes a reaction that results in thedegradation of a undesirable, toxic and/or cardiotoxic sphingolipid, ora metabolite thereof; and such an enzyme may be one that has beenaltered via molecular genetics to have improved desirable propertiessuch as enhanced catalytic activity, tighter substrate binding, etc.

[0043] In another version of this form of sphingolipid-based therapy,the therapeutic agent is a nucleic acid (including, without limitation,DNA, RNA, and oligonucleotides). A therapeutic nucleic acid may have asequence that is antisense to a nucleotide sequence found within an mRNAthat encodes an enzyme that catalyzes a reaction that results in theproduction of a undesirable, toxic and/or cardiotoxic sphingolipid, or ametabolite thereof, or a receptor thereof. Such nucleic moleculesinclude antisense oligonucleotides. Such antisense nucleic acids bind toa specific target mRNA due to their complementary sequences, and preventthe mRNA from being processed or translated, or enhance or cause thedegradation of the mRNA. A therapeutic nucleic acid may be a genetherapy construct that comprises and expresses, over-expresses orconstitutively expresses (i) nucleic acids that are antisense to thosethat encode an enzyme that catalyzes a reaction that results in theproduction of a undesirable, toxic and/or cardiotoxic sphingolipid or ametabolite thereof; (ii) therapeutic proteins, such as enzyme thatdegrades a sphingolipid, or a dominant negative mutant that inhibitssuch an enzyme, or a sphingolipid-binding protein.

[0044] Any composition and method of the invention that may be used insphingolipid-based therapy may be used in combination with any othercompositions and methods for sphingolipid-based therapy, as well as inconjunction with therapeutic agents and compositions that are notsphingolipid-based. Useful adjuvant treatments for thesphingolipid-based treatments of the invention modulate thesphingomyelin signaling pathway and/or inhibit cytokines (see theDetailed Description and Example14). An “adjuvant” is any agent that isadded to a composition or therapeutic regimen to aid the therapeuticeffect of the active agent(s) thereof.

[0045] An agent for sphingolipid-based therapy is formulated in apharmaceutical composition. The pharmaceutical compositions of theinvention may be formulated for rapid cardiac delivery. By “rapidcardiac delivery” it is meant that the therapeutic agent reaches atherapeutically effective concentration in the blood, serum, orspecified tissue within about 30 to 60 minutes, preferably within about15 to 20 minutes, more preferably within about 5 to 10 minutes, and mostpreferably within about 5 seconds to about 5 minutes, after itsadministration. The pharmaceutical compositions are used to treatcardiac, myocardial and other diseases, disorders or physical trauma.

[0046] Pharmaceutical and pharmaceutical compositions comprising one ormore therapeutic agents of the invention are incorporated into kits andmedical devices for such treatments. Medical devices are used toadminister the pharmaceutical compositions of the invention to a patientin need thereof, and kits that include such devices. Such devices andkits may be designed for the routine administration, includingself-administration, of the pharmaceutical compositions of theinvention. Such devices and kits may also be designed for emergency use,i.e., in ambulances or emergency rooms, or during surgery, or inactivities where injury is possible but where fall medical attention maynot be immediately forthcoming (i.e., hiking and camping, or combatsituations). The invention thus provides cardiac and myocardialtherapies based on the role of sphingolipids in cardiac and myocardialdiseases, disorders and physical trauma.

[0047] The invention also provides screening assays, includinghigh-throughput screening (HTS) assays, that are useful for identifyingnovel sphingolipid-based therapeutics. Chemical libraries are screenedusing these assays, preferably in a high throughput manner, to identifylead compounds and therapeutic agents.

[0048] In a related aspect, the invention provides a method ofidentifying molecules that specifically bind to, and/or otherwiseinterfere with the action of a sphingolipid target. A “sphingolipidtarget” is any molecule or moiety that is desired to obtain novelcompounds that bind thereto or otherwise inhibit the activity thereof.Sphingolipid targets of the inventions include, but are not limited to,sphingolipids per se; sphingolipid receptors; and molecules involved insphingolipid metabolism, including but not limited to enzymes that acton sphingolipids and sphingolipid metabolites.

[0049] In another embodiment, sphingolipids that are cardiotoxic atrelatively high concentrations are used to preconditon hearts.Preconditioning hearts with short cycles of ischemia and reperfusion isknown to have a cardioprotective effect in rodents (Yellon et al.,Cardiovasc Res 26:983-987, 1992; Napoli et al., J Clin Bas Cardiol1:37-42, 1998). In the preconditioning methods of the invention,sphingolipids that are cardiotoxic are administered in small doses. Inthe methods of the invention, sphingolipids, including but not limitedto ceramide, sphingosine and sphingosine-1-phosphate given in low,intermittent doses may protect cardiac tissue from ischemia.

[0050] The invention provides benefits not previously obtainable incardiovascular, cardiac and myocardial treatments. By way ofnon-limiting example, the consequences of acute cardiac or myocardialevents may result from the end result of a cascade of molecular eventsthat evolve rapidly after symptoms become apparent. Treatments thataddress early events in the cascade may not be able to “catch up” withsuch events, i.e., may not achieve an effective level until after someundesirable molecules that lead to cardiac or myocardial damage havebeen produced. Sphingolipid-based therapies act on undesirable eventsand molecules that occur or are present at the later stages of thesecascades, they can act before such undesirable events occur orundesirable molecules are produced, and thus can prevent the occurrenceof such events and/or production of such compounds to a greater degreethan can be realized by therapies that act earlier in the cascade.Sphingolipid-based therapies addresses events that lead directly (ratherthan indirectly) to myocardial ischemia and other cardiac disorders, andundesirable side-effects of indirect treatments are thus reduced,minimized or eliminated. Sphingolipid-based therapies provide forpreventative treatments that achieve an effective state relativelyquickly and non-intrusive as compared to other preventative measures,e.g., changes in diet or surgery.

[0051] The summary of the invention described above is not limiting andother features and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, as well asfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 shows a set of biochemical reactions that are a centralpart of the sphingolipid metabolic pathways.

[0053]FIG. 2 is a more expansive view of sphingolipid metabolism andincludes the biochemical reactions and enzymes shown in FIG. 1.Abbreviations: DAG, diacylglycerol); PtdCho, phosphatidylcholine.

[0054]FIG. 3 shows the fate of ³H-labeled SPH in whole blood. Afterlabeled sphingosine (SPH) is added to human whole blood, theconcentration of SPH drops while the concentration of labeledsphingosine-1-phosphate (S1P) increases, suggesting that SPH isconverted into S-1-P in blood; in contrast, little of the label isdetected as labeled hexadecanal (HD). Symbols: solid line, S1P; linewith long dashes, HD; line with short dashes, SPH.

[0055]FIG. 4 shows results of experiments that demonstrate thatL-carnitine blocks the hypoxia-induced production of sphingosine in acellular model.

[0056]FIG. 5 shows results of experiments in which rat hearts aresubject to ischemia with (grey lines) or without (black lines) aninhibitor of sphingomyelinase.

[0057]FIG. 6 shows the general chemical structure of aminoglycosides.“R1” through “R13” are substituent groups.

ABBREVIATIONS

[0058] Unless otherwise indicated, the following abbreviations are usedherein. Sphingolipids DHSPH Dihydrosphingosine CER ceramide(N-acylsphingosine) SPC Sphingosylphosphorylcholine SPH SphingosineS-1-P sphingosine-1-phosphate (a.k.a. S1P or SPP) SM SphingomyelinEnzymes CER kinase ceramide kinase SMase Sphingomyelinase SM-deacylaseSphingomyelin deacylase SPH kinase Sphingosine kinase S-1-P LyaseSphingosine-1-phosphate lyase S-1-P Sphingosine-1-phosphate lyasephosphatase Phosphatase

DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention provides methods and compositions usefulfor the treatment of cardiovascular and cerebrovascular diseases anddisorders, as well as other disease states that relate to sphingolipidsand sphingolipid metabolites. The methods and compositions act byinterfering with the metabolism of various sphingolipids and/or theirmetabolites; by binding sphingolipids, thereby reducing their effectiveconcentration; by modulating the sphingomyelin signaling pathway; viamodalities based on molecular genetics (including but not limited to theuse of dominant negative proteins, antisense, gene therapy, and thelike). All the above modalities of cardiovascular therapy may be usedalone, in combination with each other, and/or in combination with othermethods and compositions useful for cardiovascular therapy (includingbut not limited to those that interfere with the action of certaincytokines). The therapeutic methods and compositions of the inventionare said to be “sphingolipid-based” in order to indicate that thesetherapies act by changing the relative, absolute or availableconcentration(s) of certain undesirable, toxic or cardiotoxicsphingolipids. Therapeutic adminstration of exogenous sphingolipids mayhave therapeutic benefit if give in a preconditiong regimen (i.e., lowdoses given intermittently).

[0060] Applicants believe, without wishing to be bound by any particulartheory, that the level of undesirable sphingolipids such as CER, SPH orS-1-P, and/or one or more of their metabolites, may be directlyresponsible for cardiac dysfunction, during or immediately after cardiacischemia such as during reperfusion injury. For example, sphingosine hasnegative inotropic effects on the heart (Oral H, Dom G. W., Mann D. L.Sphingosine mediates the immediate negative inotropic effects of tumornecrosis factor-a in the adult mammalian cardiac myocyte. J. Biol. Chem.1997;272:4836-4842; Krown K, Yasui K, Brooker M, et al. TNFα receptorexpression in rat cardiac myocytes: TNFα inhibition of L-typeCa2+current and Ca2+transients. FEBS Letters 1995;376:24-30; Smith G.W., Constable P. D., Eppley R. M., Tumbleson M. E., Gumprecht L. A.,Haschek-Hock W. M. Purified fumonisin B1 decreases cardiovascularfunction but does not alter pulmonary capillary permeability in swine.Toxicological Sciences 2000;56:240-249, blocks Na/Ca exchangersCondrescu M, Reeves J. P. Inhibition of sodium-calcium exchange byceramide and sphingosine. J. Biol. Chem. 2001;276:4046-4054, and theL-type calcium channel in heart cells Krown K, Yasui K, Brooker M, etal. TNFa receptor expression in rat cardiac myocytes: TNFa inhibition ofL-type Ca2+current and Ca2+transients. FEBS Letters 1995;376:24-30, andmodulates the ryanodine receptor McDonough P. M., Yasui K, Betto R, etal. Control of cardiac Ca2+levels: inhibitory actions of sphingosine onCa2+transients and L-channel conductance. Circ. Res. 1994;75:981-989,all of which can cause calcium deregulation that is observed whencardiac cells are treated with sphingosine (McDonough P. M., Yasui K,Betto R, et al. Control of cardiac Ca2+levels: inhibitory actions ofsphingosine on Ca2+transients and L-channel conductance. Circ. Res.1994;75:981-989; Krown K, Yasui K, Brooker M, et al. TNFα receptorexpression in rat cardiac myocytes: TNFα inhibition of L-typeCa2+current and Ca2+transients. FEBS Letters 1995;376:24-30). Inaddition, SPH inhibits the Na/H exchanger (Lowe J. H. N., Huang C-L,Ives H. E. Sphingosine differentially inhibits activation of theNa+/H+exchange by phorbol esters and growth factors. J. Biol. Chem.1990;265:7188-7194), that is responsible for pH regulation. Sphingosinehas also been shown to produce cell death in heart cells Krown K. A,Page MT, Nguyen C, et al. TNFα-induced apoptosis in cardiac myocytes:Involvement of the sphingolipid signalling cascade in cardiac celldeath. J. Clin. Invest. 1996;98:2854-2865; Zechner D, Craig R, HanfordD, McDonough P. M., Sabbadini R. A., Glembotski C. C. MKK6 inhibitsmyocardial cell apoptosis via a p38 MAP kinase-dependent pathway. J.Biol. Chem. 1998;273:8232-8239),and can also produce free radica damageduring reperfusion Hernandez O, Discher, D., Bishorpric, N., Webster, K.Rapid Activation of Neutral Sphingomyelinase by Hypoxia-Reoxygenation ofCardiac Myocytes. Circ. Res 2000:198-204. Sphingosine-1-phosphate hasbeen shown to produe cell death Zechner D, Craig R, Hanford D, McDonoughP. M., Sabbadini R. A., Glembotski C. C. MKK6 inhibits myocardial cellapoptosis via a p38 MAP kinase-dependent pathway. J. Biol. Chem.1998;273:8232-8239, and promote arrthymias and coronary vasoconstrictionSugiyama A, Yatomi Y, Ozaki Y, Hashimoto K. Sphingosine 1-phosphateinduces sinus tachycardia and coronary vasoconstriction in the canineheart. Cardiovasc. Res. 2000;46:119-125; MacDonnell K, Severson D, GilesW. Depression of excitability by sphingosine 1-phosphate in ratventricular myocytes. Am. J. Physiol. 1998;44:H2291-H2299; and Liliom K,Sun G, Bunemanns M, et al. Sphingosylphosphocholine is a naturallyoccuring lipid mediator in blood plasma: a possible role in regulatingcardiac function via sphingolipid receptors. Biochem J.2001;355:189-197).

[0061] Because sphingolipids such as S-1-P are involved in fibrogenesisand wound healing of liver tissue (Davaille et al., J. Biol. Chem.275:34268-34633, 2000; Ikeda et al., Am J. Physiol. Gastrointest. LiverPhysiol 279:G304-G310, 2000), healing of wounded vasculatures (Lee etal., Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000), and otherdisease states, or events associated with such diseases, such as cancer,angiogenesis and inflammation (Pyne et al., Biochem. J. 349:385-402,2000), the compositions and methods of the disclosure may be applied totreat these diseases as well as cardiac diseases. For example, S-1-P maybe used therapeutically as a promoter of cardiac angiogenesis. Theability of S-1-P to stimulate angiogenesis in cell culture and innon-cardiac tissue has been reported (Lee et al., Sphingosine1-Phosphate induces angiogenesis: its angiogenic action and signalingmechanism in human umbilical endothelial cells. Biochem Biophys ResCommun 1999;264:743-325; Lee et al., Am J. Physiol Cell Physiol278:C612-C618, 2000). Recent evidence suggests that exogenouslyadministered S-1-P crosses the blood-brain barrier and promotes cerebralvasoconstriction (Tosaka et al., Stroke 32: 2913-2919. 2001). Thissuggests that sphingolipids derived from cardiac or other non-cerebralsources could contribute to stroke. Consequently, interfering withsphingolipid production and/or action may be beneficial in mitigatingstroke, particularly in stroke casued by peripherical vascular disease,atherosclerosis and cardiac disorders. s.For example, S1P may be usedtherapeutically as a promoter of cardiac angiogenesis. The ability ofS1P to stimulate angiogenesis in cell culture and in non-cardiac tissuehas been reported [Lee, 1999 #1508]. Recent evidence suggests thatexogenously administered S1P crosses the blood-brain barrier andpromotes cerebral vasoconstriction (Tosaka et al., Stroke 32: 2913-2919.2001). This suggests that sphingolipids derived from cardiac or othernon-cerebral sources could contribute to stroke. Consequently,interfering with sphingolipid production and/or action may be beneficialin mitigating stroke, particularly in stroke casued by periphericalvascular disease, atherosclerosis and cardiac disorders.

[0062] It has been suggested that an early event in the course ofcardiac ischemia (i.e., lack of blood supply to the heart) is an excessproduction by the heart muscle of the naturally occurring compoundsphingosine, and that other metabolites, particularlysphingosine-I-phosphate (S-1-P), are also produced either by the hearttissue itself or by components of blood as a consequence of cardiacsphingolipid production and subsequent conversion in the blood. Thepresent invention provides methods and the compositions thereof toinhibit and/or activate sphingolipid production and/or metabolism. Morespecifically, the present invention provides methods and thecompositions that may block production of SPH, S-1-P and othermetabolites by inhibiting and/or activating metabolic enzymes and/orsphingolipid receptors involving in the sphingolipid metabolic pathways.Since either hypoxia per se and/or cardiac-derived TNFα. and/or othercytokines may trigger the sphingomyelin signal transduction cascade inthe heart to increase the production of SPH, S-1-P and othermetabolites, the present invention also provides methods andcompositions to block cytokine release and/or its action.

[0063] The present invention thus provides methods and compositionsthereof to reduce blood and tissue levels of key sphingolipids, e.g.,SPH and S-1-P. Such methods and compositions include, but are notlimited to, monoclonal and/or polyclonal antibodies directed tosphingolipids, which may be used, for example, to bind and thus lowerthe effective concentration of, undesirable sphingolipids in wholeblood. The present invention also provides methods and the compositionsthereof to indirectly reduce the absolute or effective (available) bloodand tissue levels of key sphingolipids, e.g. SPH and S-1-P, includingbut not limited to methods and compositions for inhibiting and/oractivating enzymes involving in the sphingolipid metabolic pathways; forthe use of soluble fragments containing the sphingolipid binding domainof enzymes involved in sphingolipid metabolism, or the binding domain ofsphingolipid binding proteins, to bind and reduce the effectiveconcentration of undesirable sphingolipids; for the use of negativedominant (a.k.a. “transdominant”) mutants of sphingolipid receptors andenzymes involved in sphingolipid metabolism; for genetic therapy toprovide or alter a function of a sphingolipid enzyme or receptor; andfor the use of antisense oligonucleotides or transcripts against mRNAsof the sphingolipid metabolic enzymes, and/or sphingolipid receptors, toreduce or eliminate the genetic expression of these enzymes. For areview of sphingolipid metabolism, see Liu et al., Crit Rev. Clin. Lab.Sci. 36:511-573, 1999.

[0064] The present invention also provides compositions for inhibitingthe action or expression of cytokines, interferons, chemokines and thelike, that may modulate events that occur during the sphingomyelinsignaling pathway. This pathway, which it has been suggested isactivated during cardiac ischemia/hypoxia (Bielawska et al., Am. J.Pathol. 151:1257-1263, 1997; Meldrum, Am. J. Physiol. 274:R577-R595,1998; and Cain et al., J. Mol. Cell. Cardiol. 31:931-947, 1999), andwhich is stimulated by cytokines, interferons, chemokines and the like,ultimately results in increased ceramide production. An increased levelof ceramide would, in turn, be expected to result in elevatedconcentrations of undesirable sphingolipids such as, e.g., S-1-P andSPH. For reviews of the sphingomyelin signaling pathway, see Hannun etal., Adv. Lipid Res. 25:27-41, 1993; Liu et al., Crit. Rev. Clin. Lab.Sci. 36:511-573, 1999; Igarashi, J. Biochem. 122:1080-1087, 1997; Oralet al., J. Biol. Chem. 272:4836-4842, 1997; and Spiegel et al.,Biochemistry (Moscow) 63:69-83, 1998.

[0065] Sphingolipids

[0066] The therapeutic methods and compositions of the invention aresaid to be “sphingolipid-based” in order to indicate that thesetherapies can change the relative, absolute or availableconcentration(s) of certain undesirable, toxic or cardiotoxicsphingolipids. “Toxic sphingolipids” are those that can, under certaincircumstances, disturb the normal function of cells such as ones thatcause or enhance the necrosis and/or apoptosis of cells, including, insome instances, particular cell types that are found in specific tissuesor organs. “Cardiotoxic sphingolipids” are toxic sphingolipids thatdirectly or indirectly cause a negative inotropic state or cause orenhance the necrosis and/or apoptosis of cells found in or associatedwith the heart, including but not limited to cardiomyocytes, cardiacneurons and the like, and/or can cause loss of cardiac function due tothe negative inotropic, arrhythmic coronary vasoconstriction/spasmeffects of the sphingolipids and/or their metabolites. “Undesirablesphingolipids” include toxic and cardiotoxic sphingolipids, as well asmetabolites, particularly metabolic precursors, of toxic and cardiotoxicsphingolipids. Undesirable sphingolipids of particular interest includebut are not limited to ceramide (CER), sphingosine-1-phosphate (S-1-P),and sphingosine (SPH).

[0067] Sphingolipids are a unique class of lipids that were named, dueto their initially mysterious nature, after the Sphinx. Sphingolipidswere initially characterized as primary structural components of cellmembranes, but recent studies indicate that sphingolipids also serve ascellular signaling and regulatory molecules (Hannun et al., Adv. LipidRes. 25:27-41, 1993; Speigel et al., FASEB J. 10:1388-1397, 1996;Igarashi, J. Biochem 122:1080-1087, 1997). The metabolic pathways forsphingolipids are shown in FIGS. 1 and 2.

[0068] One group of sphingolipids of particular interest is the set ofsphingolipids involved in the sphingomyelin signal transduction pathway(Hannun et al., Adv. Lipid Res. 25:27-41, 1993; Liu et al., Crit. Rev.Clin. Lab. Sci. 36:511-573, 1999). In this regard, ceramide, sphingosineand sphingosine-l-phosphate have been most widely studied (Hannun etal., Science 243:500-507, 1989). Sphingolipid signaling molecules arederived from sphingomyelin and include but are not limited tosphingosine [(SPH; D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol orsphingenine)], sphingosine-1-phosphate (S-1-P), ceramide (CER), as wellas sphingosylphosphorylcholine (SPC) (see FIG. 1).

[0069] Ceramide and sphingomyelin (SPH) are intracellular secondmessengers activated by the sphingomyelin signal transduction cascadethat occurs in response to inflammatory cytokines such as TNFα, γIFN,and IL-1β, and in response to ischemia/reperfusion (Bielawska et al.,Am. J. Pathol. 151:1257-1263, 1997; Zager et al., Kidney Int. 54:60-70,1997).

[0070] Accumulations of ceramide in ischemia of human and rat brains,and in renal ischemia have been alleged to occur (Kubota et al., JapanJ. Exp. Med. 59:59-64, 1989; Kubota et al., Neuro. Res. 18:337-341,1996; and Zager et al., Kidney Int. 54:60-70, 1997). Further, S1P causescerebral vasoconstriction (Tosaka et al., Stroke 32: 2913-2919. 2001).Taken together, it is reasoned that either brain-derived or non-brainderived sphingolipids may contribute to stroke and that interfering withsphingolipid production and/or action may mitigate stroke.

[0071] Hernandez et al. (Circ. Res. 86:198-204, 2000) is stated to showthat one of the earliest responses of cardiac myocytes to hypoxia andreoxygenation is the activation of neutral sphingomyelinase and theaccumulation of ceramide. SPH has been allegedly implicated as mediatingan early signaling event in apoptotic cell death in a variety of celltypes (Ohta et al., FEBS Letters 355:267-270, 1994; Ohta et al., CancerRes. 55:691-697, 1995; Cuvlilier et al., Nature 381:800-803, 1996). Itis postulated that the cardiotoxic effects of hypoxia may result in partfrom sphingolipid production and/or from the inappropriate production ofother metabolites (e.g. protons, calcium, certain free radicals) orsignaling molecules (e.g., MAP kinases, caspases) that adversely affectcardiac function.

[0072] S-1-P is stored in platelets and is a normal constituent of humanplasma and serum (Yatomi et al., J. Biochem. 121:969-973, 1997).Sugiyama et al. (Cardiovascular Res. 46:119-125, 2000) is stated todemonstrate that S-1-P is a coronary vasoconstrictor and has otherbiological effects on canine hearts. Siess et al. have proposed a rolefor S-1-P in artherosclerosis (IUBMB Life 49:161-171, 2000). This hasbeen supported by other data, including evidence that the protectiveeffect of HDL is due to blocking S1P production (Xia et al., PNAS95:14196-14201, 1988; Xia et al., J. Biol Chem 274:33143-33147, 1999).

[0073] Treatment of neonatal and adult cardiac cells in culture withphysiologically relevant levels of SPH and its immediate metabolite,S-1-P, has been related to the activation of cardiomyocyte cell death byapoptosis, a form of programmed cell death that may contribute to thesize of the size of myocardial infarct (Krown et al., J. Clin. Invest.98:2854-2865, 1996; Zechner et al., J. Biol. Chem. 273:8232-8239, 1998;Kajstura et al., Lab. Invest. 74:86-107, 1996).

[0074] Cordis et al. (J. Pharm. and Biomed. Analysis 16:1189-1193, 1998)states that levels of sphingosine are reduced in ischemia/reperfused rathearts. In contrast, however, Bielawska et al., Am. J. Pathol.151:1257-1263, 1997) is stated to present evidence that the levels ofthe immediate metabolic precursor of SPH, ceramide, are increased in ratneonatal cardiomyocytes perfused under ischemic conditions.

[0075] Sphingomyelin, the metabolic precursor of ceramide, has beenstated to be increased in experimental animals subjected to hypoxia(Sergeev et al., Kosm. Biol. Aviakosm. Med. (Russian) 15:71-74, 1981).Other studies have been stated to show that internal membranes of musclecells contain high amounts of SPH and sphingomyelin (Sumnicht et al.,Arch. Biochem. Biophys. 215:628-637, 1982; Sabbadini et al., Biochem.Biophys. Res. Comm. 193752-758, 1993). Treatment of experimental animalswith fumonisinB fungal toxins result in increase serum levels of SPH andDHSPH (S1P was not measured) with coincident negative inotropic effectson the heart (Smithe et al., Toxicological Sciences 56:240-249, 2000).

[0076] Modulation of the Metabolism of Sphingolipids for TherapeuticBenefit

[0077] One way to control the amount of undesirable sphingolipids in apatient is to alter the activity of an enzyme that catalyzes a reactionthat is part of sphingolipid metabolism (see FIGS. 1 and 2).Specifically, to lower the amount of undesirable sphingolipids, one caninhibit or block enzymes involved in sphingolipid anabolism(constructive metabolism, i.e., reactions that lead to the production ofundesirable sphingolipids). Additionally or alternatively, one canstimulate or activate enzymes involved in sphingolipid catabolism(destructive metabolism, i.e., reactions that lead to the breakdown ofundesirable sphingolipids). For further details, see Examples 7-10.

[0078] There are several enzymes involved in the sphingolipid metabolicpathway that can be inhibited in order to reduce the amount ofundesirable sphingolipids. As is explained herein, due to theirdeleterious effects on cardiac cells and tissues, two particularlyundesirable sphingolipids are SPH and S-1-P. Enzymes that are inhibitedfor the purpose of lowering levels of SPH, S-1-P and/or otherundesirable sphingolipids are assigned to different classes based on theproduct(s) of the reaction that they catalyze.

[0079] Similarly, there are several enzymes involved in sphingolipidmetabolism that can be stimulated in order to reduce the amount ofundesirable sphingolipids including but not limited to SPH and S-1-P.Stimulation of these enzymes leads to a more rapid degradation ofundesirable sphingolipids. Enzymes that are stimulated for the purposeof lowering levels of SPH, S-1-P and other undesirable sphingolipids areassigned to different classes based on whether they promote theproduction or degradation of a selected undesirable, toxic and/orcardiotoxic sphingolipid or a precursor thereof. In general, enzymesthat catalyze the production of a undesirable, toxic and/or cardiotoxicsphingolipid or its precursor are inhibited, whereas enzymes thatcatalyze the degradation of the undesirable, toxic and/or cardiotoxicsphingolipid are stimulated.

[0080] Binding Sphingolipids, and Receptors Thereof, for TherapeuticBenefit

[0081] One way to control the amount of undesirable sphingolipids in apatient is by providing a composition that binds one or moresphingolipids, or receptors thereof.

[0082] Antibodies and other compounds that bind to undesirablesphingolipids may be used as therapeutic “sponges” that reduce the levelof free undesirable sphingolipids. When a compound is stated to be“free,” the compound is not in any way restricted from reaching the siteor sites where it exerts its undesirable effects. Typically, a freecompound is present in the cardiovascular system, which either is orcontains the site(s) of action of the free compound, or from which acompound can freely migrate to its site(s) of action. A free compoundmay also be available to be acted upon by any enzyme that converts thecompound into an undesirable compound.

[0083] Antibodies and other compounds that bind to cellular receptors ofundesirable sphingolipids may be used to compete with and/or preventsphingolipids from binding to receptors and thereby causing or enhancingundesirable cellular or biochemical events. Such events include, but arenot limited to, the entry of undesirable sphingolipids into cells,initiation of a signal cascade pathway that has an undesirable outcome,and a reaction, which may be catalyzed by an enzyme, that produces anundesirable product. Receptors of interest include but are not limitedto Edg receptors, SCaMPER. and other receptors that bind sphingolipids,and receptors for cytokines, including but not limited to the TNFαreceptor.

[0084] Antibodies

[0085] Several antibodies have recently been approved for therapeuticuse in humans by the Federal Drug Administration (Kling, Mod. Drug Disc.2:33-45, 1999). In one aspect of sphingolipid-based cardiovasculartherapy, antibodies that bind sphingolipids can be delivered to apatient, e.g., incorporation into pharmaceutical compositions, medicaldevices, and the like, for use in sphingolipid-based cardiovasculartherapy. Such methods may, by way of non-limiting example, (1) modulatethe effective concentration of a undesirable, toxic and/or cardiotoxicsphingolipid or a metabolic precursor thereof; (2) sterically inhibitthe binding of a sphingolipid to a cellular receptor therefor, or tolower the concentration of a sphingolipid that is available for bindingto such a receptor; (3) sterically inhibit the enzymatic conversion of ametabolic precursor of a undesirable, toxic and/or cardiotoxicsphingolipid, or lower the concentration of such a precursor that isavailable for enzymatic conversion into a undesirable, toxic and/orcardiotoxic sphingolipid; and (4) remove undesirable, toxic and/orcardiotoxic sphingolipids and their metabolic precursors from blood invivo or ex vivo.

[0086] The term “antibody” is meant to encompass an immunoglobulinmolecule obtained by in vitro or in vivo generation of an immunogenicresponse, and includes polyclonal, monospecific and monoclonalantibodies, as well as T cell receptors, and fragments and derivativesthereof. An “immunogenic response” is one that results in the productionof antibodies directed to one or more proteins after the appropriatecells have been contacted with such proteins, or polypeptide derivativesthereof, in a manner such that one or more portions of the proteinfunction as epitopes. An epitope is a single antigenic determinant in amolecule. In proteins, particularly denatured proteins, an epitope istypically defined and represented by a contiguous amino acid sequence.However, in the case of nondenatured proteins, epitopes also includestructures, such as active sites, that are formed by thethree-dimensional folding of a protein in a manner such that amino acidsfrom separate portions of the amino acid sequence of the protein arebrought into close physical contact with each other.

[0087] Polyclonal antibodies are generated in a immunogenic response toa protein having many epitopes, and thus include a variety of differentantibodies directed to different epitopes within the protein. Methodsfor producing polyclonal antibodies are known in the art (see, e.g.,Cooper et al., Section III of Chapter 11 in: Short Protocols inMolecular Biology, 2nd Ed., Ausubel et al, eds., John Wiley and Sons,New York, 1992, pages 11-37 to 11-41).

[0088] Monospecific antibodies (a.k.a. antipeptide antibodies) aregenerated in a humoral response to a short (typically, 5 to 20 aminoacids) immunogenic polypeptide that corresponds to a few (preferablyone) isolated epitopes of the protein from which it is derived. Aplurality of monospecific antibodies includes a variety of differentantibodies directed to a specific portion of the protein, i.e., to anamino acid sequence that contains at least one, preferably only one,epitope. Methods for producing monospecific antibodies are known in theart (see, e.g., Id., pages 11-42 to 11-46).

[0089] A monoclonal antibody is a specific antibody that recognizes asingle specific epitope of an immunogenic protein. In a plurality of amonoclonal antibody, each antibody molecule is identical to the othersin the plurality. In order to isolate a monoclonal antibody, a clonalcell line that expresses, displays and/or secretes a particularmonoclonal antibody is first identified; this clonal cell line can beused in one method of producing the antibodies of the invention. Methodsfor the preparation of clonal cell lines and of monoclonal antibodiesexpressed thereby are known in the art (see, for example, Fuller et al.,Section II of Chapter 11 in: Short Protocols in Molecular Biology, 2ndEd., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages11-22 to 11-11-36).

[0090] T cell receptors (TCR) are a distinct class of proteins that aregenetically and structurally related to antibodies. TCR proteins belongto the immunoglobulin superfamily of proteins and have molecularstructures similar to those of antibodies and, like antibodies,specifically recognize (i.e., specifically and bind) specific ligands.Complexes of TCR are displayed on T cells and bind specific antigens forthe purpose of triggering molecular events associated with T celldifferentiation and activation. Like antibodies, TCR proteins recognizeparticular antigens. However, because of differences in the precisestructures of the portions of TCR proteins that bind ligands and theamino acid sequences associated with those structures, as well asdifferent mechanisms by which genes encoding a protein are diversifiedby rearrangement and mutation. Thus, the “molecular rules” for specificbinding of TCR molecules to their ligands are different from those ofantibodies, and the use of TCR proteins expands the population ofpotential sphingolipid-binding proteins.

[0091] Antibody fragments and derivatives are proteins that are derivedfrom antibodies and T-cell receptors and which retain the ability tospecifically recognize the ligand recognized by the “parent” antibody orTCR (see Gavilondo et al., BioTechniques 29:128-145, 2000, and Morrow,Amer. Lab. 32:15-19, 2000). Preferred fragments include Fab fragments(i.e., an antibody fragment that contains the antigen-binding domain andcomprises a light chain and part of a heavy chain bridged by a disulfidebond); Fab′ (an antibody fragment containing a single anti-bindingdomain comprising an Fab and an additional portion of the heavy chainthrough the hinge region); F(ab′)2 (two Fab′ molecules joined byinterchain disulfide bonds in the hinge regions of the heavy chains; theFab′ molecules may be directed toward the same or different epitopes); abispecific Fab (an Fab molecule having two antigen binding domains, eachof which may be directed to a different epitope); and camelized VHdomains (the variable, antigen-binding determinative region of a singleheavy chain of an antibody in which some amino acids at the VH interfaceare those found in the heavy chain of naturally occurring camelantibodies).

[0092] Single chain antibodies (scFv) comprise a variable region,a.k.a., a scFv (the variable, antigen-binding determinative region of asingle light and heavy chain of an antibody linked together by a chainof 10-25 amino acids). For reviews, see Raag et al., Single-chain Fvs.FASEB J. 9:73-80, 1995, and Hudson, Recombinant antibody fragments.Curr. Op. Biotechnol. 9, 395-402, 1999. See also Bird et al.,Single-chain antigen-binding proteins. Science 242, 423-426, 1988, andU.S. Pat. Nos. 5,260,203; 5,869,620; 5,455,030; 5,518,889; 5,534,621;4,946,778; 6,025,165; and 6,027,725.

[0093] The well-known technique of phage display is used to prepare scFvmolecules. For reviews, see Winter et al., Making antibodies by phagedisplay technology. Annu. Rev. Immunol. 12:433-455, 1994; Little et al.,Surface display of antibodies. Biotechn. Adv. 12:539-555, 1994; andBurton et al., Human antibodies from combinatorial libraries. Adv.Immunol. 57:191-280, 1994. See also U.S. Pat. Nos. 5,821,047; 5,702,892;6,031,071; and 6,310,191.

[0094] Complexes of single chain antibodies are also within the scope ofthe invention and include, but are not limited to, a disulfide-linkedFv, or dsFv (the variable, antigen-binding determinative region of asingle light and heavy chain of an antibody linked together by adisulfide bond; a bispecific sFv (a scFv or a dsFv molecule having twoantigen-binding domains, each of which may be directed to a differentepitope); a diabody (a dimerized scFv formed when the VH domain of afirst scFv assembles with the VL domain of a second scFv and the VLdomain of the first scFv assembles with the VH domain of the secondscFv; the two antigen-binding regions of the diabody may be directedtowards the same or different epitopes); and a triabody (a trimerizedsFv, formed in a manner similar to a diabody, but in which threeantigen-binding domains are created in a single complex; the threeantigen binding domains may be directed towards the same or differentepitopes).

[0095] The term “antibody” also includes genetically engineeredantibodies and/or antibodies produced by recombinant DNA techniques and“humanized” antibodies. Humanized antibodies have been modified, bygenetic manipulation and/or in vitro treatment to be more human, interms of amino acid sequence, glycosylation pattern, etc., in order toreduce the antigenicity of the antibody or antibody fragment in ananimal to which the antibody is intended to be administered (Gussow etal., Methods Enz. 203:99-121, 1991).

[0096] Methods of Preparing Antibodies and Antibody Variants

[0097] The antibodies and antibody fragments of the invention may beproduced by any suitable method, for example, in vivo (in the case ofpolyclonal and monospecific antibodies), in cell culture (as istypically the case for monoclonal antibodies, wherein hybridoma cellsexpressing the desired antibody are cultured under appropriateconditions), in in vitro translation reactions, and in recombinant DNAexpression systems (Johnson et al., Methods Enz. 203:88-98, 1991).Antibodies and antibody variants can be produced from a variety ofanimal cells, preferably from mammalian cells, with murine and humancells being particularly preferred. Antibodies that includenon-naturally occurring antibody and T-cell receptor variants thatretain only the desired antigen targeting capability conferred by anantigen binding site(s) of an antibody can be produced by known cellculture techniques and recombinant DNA expression systems (see, e.g.,Johnson et al., Methods in Enzymol. 203:88-98, 1991; Molloy et al., Mol.Immunol. 32:73-81, 1998; Schodin et al., J. Immunol. Methods 200:69-77,1997). Recombinant DNA expression systems are typically used in theproduction of antibody variants such as, e.g., bispecific antibodies andsFv molecules. Preferred recombinant DNA expression systems includethose that utilize host cells and expression constructs that have beenengineered to produce high levels of a particular protein. Preferredhost cells and expression constructs include Escherichia coli; harboringexpression constructs derived from plasmids or viruses (bacteriophage);yeast such as Sacharomyces cerevisieae or Fichia pastoras harboringepisomal or chromosomally integrated expression constructs; insect cellsand viruses such as Sf9 cells and baculovirus; and mammalian cellsharboring episomal or chromosomally integrated (e.g., retroviral)expression constructs (for a review, see Verma et al., J. Immunol.Methods 216:165-181, 1998). Antibodies can also be produced in plants(U.S. Pat. No. 6,046,037; Ma et al., Science 268:716-719, 1995) or byphage display technology (Winter et al., Annu. Rev. Immunol. 12:433-455,1994).

[0098] XenoMouse strains are genetically engineered mice in which themurine IgH and Igk loci have been functionally replaced by their Igcounterparts on yeast artificial YAC transgenes. These human Igtransgenes can carry the majority of the human variable repertoire andcan undergo class switching from IgM to IgG isotypes. The immune systemof the xenomouse recognizes administered human antigens as foreign andproduces a strong humoral response. The use of XenoMouse in conjunctionwith well-established hybridomas techniques, results in fully human IgGmAbs with sub-nanomolar affinities for human antigens (see U.S. Pat.Nos. 5,770,429, entitled “Transgenic non-human animals capable ofproducing heterologous antibodies”; 6,162,963, entitled “Generation ofXenogenetic antibodies”; 6,150,584, entitled “Human antibodies derivedfrom immunized xenomice”; 6,114,598, entitled Generation of xenogeneicantibodies; and 6,075,181, entitled “Human antibodies derived fromimmunized xenomice”; for reviews, see Green, Antibody engineering viagenetic engineering of the mouse: XenoMouse strains are a vehicle forthe facile generation of therapeutic human monoclonal antibodies, J.Immunol. Methods 231:11-23, 1999; Wells, Eek, a XenoMouse: Abgenix,Inc., Chem Biol 2000 August;7(8):R185-6; and Davis et al., Transgenicmice as a source of fully human antibodies for the treatment of cancerCancer Metastasis Rev 1999;18(4):421-5).

[0099] Soluble Receptor Fragments

[0100] Soluble polypeptides derived from membrane bound, typicallyhydrophobic, sphingolipid receptors that retain the receptor's abilityto bind sphingolipids may also be used to bind sphingolipids andsphingolipid metabolites. In the case of Edg receptors, in someinstances, particualr amino acid residues may be involved in thespecificity of sphingolipid binding, i.e., the amino acids thatdetermine which sphingolipid is bound by a specific receptor (Parrill etal., “Identification of Edg1 Receptor Residues That RecognizeSphingosine 1-Phosphate”, J. Biol. Chem. 275:39379-39384, 2000; and Wanget al., “A Single Amino Acid Determines Lysophospholipid Specificity ofthe S1P1(EDG1) and LPA1 (EDG2) Phospholipid Growth Factor Receptors”,JBC Papers in Press Published Oct. 16, 2001 as Manuscript M107301200).Such information may be used to provide soluble receptor fragmentscomprising receptor residues of interest, i.e., the stretches of aminoacids that bind the sphingolipid. Soluble receptor fragments derivedfrom the naturally soluble TNFα receptor have been prepared and at leastone of these, ENBREL® (Etanercept) is in development as a therapeuticagent for arthritis. In addition, modification of such residues maypermit the skilled artisan to tailor the binding specificities and/oraffinity of soluble receptor fragments.

[0101] Soluble receptor fragments of particular interest include Edg-1,Edg-3, Edg-5, Edg-6 and Edg-8, all of which bind the undesirablesphingolipid sphingosine-1-phosphate (S-1-P). The Edg-1, Edg-3, Edg-5receptors are of particular interest because binding of S-1-P theretoseems to stimulate the production of intracellular S-1-P (Heringdorf etal., Stimulation of intracellular sphingosine-1-phosphate production byG-protein-coupled sphingosine-1-phosphate receptors, Eur J Pharmacol.414:145-54, 2001). The P2Y(2) receptor is of interest as it alsoincreases intracellular production of S-1-P (Alemany et al., Stimulationof sphingosine-1-phosphate formation by the P2Y(2) receptor in HL-60cells: Ca(2+) requirement and implication in receptor-mediated Ca(2+)mobilization, but not MAP kinase activation, Mol Pharmacol. 58:491-7,2000).

[0102] Soluble receptor fragments may be prepared in various waysincluding but not limited to proteolytic digestion of cells or cellularmembrane preparations comprising the receptor (Bartfeld et al., Activeacetylcholine receptor fragment obtained by tryptic digestion ofacetylcholine receptor from Torpedo californica, Biochem Biophys ResCommun. 89:512-9, 1979; Borhani et al., Crystallization and X-raydiffraction studies of a soluble form of the human transferrin receptor,J. Mol Biol. 218:685-9, 1991), recombinant DNA technologies (Marlovitset al., Recombinant soluble low-density lipoprotein receptor fragmentinhibits common cold infection, J. Mol Recognit. 11:49-51, 1998; Huanget al., Expression of a human thyrotrophin receptor fragment inEscherichia coli and its interaction with the hormone and autoantibodiesfrom patients with Graves' disease, J. Mol Endocrinol. 8:137-44, 1992),or by in vitro synthesis of oligopeptides.

[0103] Nucleic Acids

[0104] Traditionally, techniques for detecting and purifying targetmolecules have used polypeptides, such as antibodies, that specificallybind such targets. While nucleic acids have long been known tospecifically bind other nucleic acids (e.g., ones having complementarysequences), aptamers (i.e., nucleic acids that bind non-nucleic targetmolecules) have been disclosed. See, e.g., Blackwell et al., Science(1990) 250:1104-1110; Blackwell et al., Science (1990) 250:1149-1152;Tuerk et al., Science (1990) 249:505-510; Joyce, Gene (1989) 82:83-87;and U.S. Pat. No. 5,840,867 entitled “Aptamer analogs specific forbiomolecules”.

[0105] As applied to aptamers, the term “binding” specifically excludesthe “Watson-Crick”-type binding interactions (i.e., A:T and G:Cbase-pairing) traditionally associated with the DNA double helix. Theterm “aptamer” thus refers to a nucleic acid or a nucleic acidderivative that specifically binds to a target molecule, wherein thetarget molecule is either (i) not a nucleic acid, or (ii) a nucleic acidor structural element thereof that is bound through mechanisms otherthan duplex- or triplex-type base pairing. Such a molecule is called a“non-nucleic molecule” herein.

[0106] Structures of Nucleic Acids

[0107] “Nucleic acids,” as used herein, refers to nucleic acids that areisolated a natural source; prepared in vitro, using techniques such asPCR amplification or chemical synthesis; prepared in vivo, e.g., viarecombinant DNA technology; or by any appropriate method. Nucleic acidsmay be of any shape (linear, circular, etc.) or topology(single-stranded, double-stranded, supercoiled, etc.). The term “nucleicacids” also includes without limitation nucleic acid derivatives such aspeptide nucleic acids (PNA's) and polypeptide-nucleic acid conjugates;nucleic acids having at least one chemically modified sugar residue,backbone, internucleotide linkage, base, nucleoside, or nucleotideanalog; as well as nucleic acids having chemically modified 5′ or 3′ends; and nucleic acids having two or more of such modifications. Notall linkages in a nucleic acid need to be identical.

[0108] Nucleic acids that are aptamers are often, but need not be,prepared as oligonucleotides. Oligonucleotides include withoutlimitation RNA, DNA and mixed RNA-DNA molecules having sequences oflengths that have minimum lengths of 2, 4, 6, 8, 10, 11, 12, 13, 14, 15,17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and maximum lengths ofabout 100, 75, 50, 40, 25, 20 or 15 or more nucleotides, irrespectively.In general, a minimum of 6 nucleotides, preferably 10 nucleotides, morepreferably 14 to 20 nucleotides, is necessary to effect specificbinding.

[0109] In general, the oligonucleotides may be single-stranded (ss) ordouble-stranded (ds) DNA or RNA, or conjugates (e.g., RNA moleculeshaving 5′ and 3′ DNA “clamps”) or hybrids (e.g., RNA:DNA pairedmolecules), or derivatives (chemically modified forms thereof). However,single-stranded DNA is preferred, as DNA is often less labile than RNA.Similarly, chemical modifications that enhance an aptamer's specificityor stability are preferred.

[0110] Chemical Modifications of Nucleic Acids

[0111] Chemical modifications that may be incorporated into aptamers andother nucleic acids include, with neither limitation nor exclusivity,base modifications, sugar modifications, and backbone modifications.

[0112] Base modifications: The base residues in aptamers may be otherthan naturally occurring bases (e.g., A, G, C, T, U, 5MC, and the like).Derivatives of purines and pyrimidines are known in the art; anexemplary but not exhaustive list includes aziridinylcytosine,4-acetylcytosine, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine (5MC), N6-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid, and 2,6-diaminopurine. In addition to nucleicacids that incorporate one or more of such base derivatives, nucleicacids having nucleotide residues that are devoid of a purine or apyrimidine base may also be included in aptamers.

[0113] Sugar modifications: The sugar residues in aptamers may be otherthan conventional ribose and deoxyribose residues. By way ofnon-limiting example, substitution at the 2′-position of the furanoseresidue enhances nuclease stability. An exemplary, but not exhaustivelist, of modified sugar residues includes 2′ substituted sugars such as2′-O-methyl-, 2′-O-alkyl, 2′-O-allyl, 2′-S-alkyl, 2′-S-allyl,2′-fluoro-, 2′-halo, or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside, ethylriboside or propylriboside.

[0114] Backbone modifications: Chemically modified backbones include, byway of non-limiting example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Chemicallymodified backbones that do not contain a phosphorus atom have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages, including without limitation morpholinolinkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl andthioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; and amide backbones.

[0115] Preparation and Identification of Aptamers

[0116] In general, techniques for identifying aptamers involveincubating a preselected non-nucleic target molecule with mixtures (2 to50 members), pools (50 to 5,000 members) or libraries (50 or moremembers) of different nucleic acids that are potential aptamers underconditions that allow complexes of target molecules and aptamers toform. By “different nucleic acids” it is meant that the nucleotidesequence of each potential aptamer may be different from that of anyother member, that is, the sequences of the potential aptamers arerandom with respect to each other. Randomness can be introduced in avariety of manners such as, e.g., mutagenesis, which can be carried outin vivo by exposing cells harboring a nucleic acid with mutagenicagents, in vitro by chemical treatment of a nucleic acid, or in vitro bybiochemical replication (e.g., PCR) that is deliberately allowed toproceed under conditions that reduce fidelity of replication process;randomized chemical synthesis, i.e., by synthesizing a plurality ofnucleic acids having a preselected sequence that, with regards to atleast one position in the sequence, is random. By “random at a positionin a preselected sequence” it is meant that a position in a sequencethat is normally synthesized as, e.g., as close to 100% A as possible(e.g., 5′-C-T-T-A-G-T-3′) is allowed to be randomly synthesized at thatposition (C-T-T-N-G-T, wherein N indicates a randomized position where,for example, the synthesizing reaction contains 25% each of A,T,C and G;or x % A, w % T, y % C and z % G, wherein x+w+y+z=100. In later stagesof the process, the sequences are increasingly less randomized andconsensus sequences may appear; in any event, it is preferred toultimately obtain an aptamer having a unique nucleotide sequence.

[0117] Aptamers and pools of aptamers are prepared, identified,characterized and/or purified by any appropriate technique, includingthose utilizing in vitro synthesis, recombinant DNA techniques, PCRamplification, and the like. After their formation, target:aptamercomplexes are then separated from the uncomplexed members of the nucleicacid mixture, and the nucleic acids that can be prepared from thecomplexes are candidate aptamers (at early stages of the technique, theaptamers generally being a population of a multiplicity of nucleotidesequences having varying degrees of specificity for the target). Theresulting aptamer (mixture or pool) is then substituted for the startingapatamer (library or pool) in repeated iterations of this series ofsteps. When a limited number (e.g., a pool or mixture, preferably amixture with less than 10 members, most preferably 1) of nucleic acidshaving satisfactory specificity is obtained, the aptamer is sequencedand characterized. Pure preparations of a given aptamer are generated byany appropriate technique (e.g., PCR amplification, in vitro chemicalsynthesis, and the like).

[0118] For example, Tuerk and Gold (Science (1990) 249:505-510) disclosethe use of a procedure termed “systematic evolution of ligands byexponential enrichment” (SELEX). In this method, pools of nucleic acidmolecules that are randomized at specific positions are subjected toselection for binding to a nucleic acid-binding protein (see, e.g., PCTInternational Publication No. WO 91/19813 and U.S. Pat. No. 5,270,163).The oligonucleotides so obtained are sequenced and otherwisecharacterization. Kinzler, K. W., et al. (Nucleic Acids Res. (1989)17:3645-3653) used a similar technique to identify syntheticdouble-stranded DNA molecules that are specifically bound by DNA-bindingpolypeptides. Ellington, A. D., et al. (Nature (1990) 346: 818-822)disclose the production of a large number of random sequence RNAmolecules and the selection and identification of those that bindspecifically to specific dyes such as Cibacron blue.

[0119] Another technique for identifying nucleic acids that bindnon-nucleic target molecules is the oligonucleotide combinatorialtechnique disclosed by Ecker, D. J. et al. (Nuc. Acids Res. 21, 1853(1993)) known as “synthetic unrandomization of randomized fragments”(SURF), which is based on repetitive synthesis and screening ofincreasingly simplified sets of oligonucleotide analogue libraries,pools and mixtures (Tuerk, C. and Gold, L. (Science 249, 505 (1990)).The starting library consists of oligonucleotide analogues of definedlength with one position in each pool containing a known analogue andthe remaining positions containing equimolar mixtures of all otheranalogues. With each round of synthesis and selection, the identity ofat least one position of the oligomer is determined until the sequencesof optimized nucleic acid ligand aptamers are discovered.

[0120] Once a particular candidate aptamer has been identified through aSURF, SELEX or any other technique, its nucleotide sequence can bedetermined (as is known in the art), and its three-dimensional molecularstructure can be examined by nuclear magnetic resonance (NMR). Thesetechniques are explained in relation to the determination of thethree-dimensional structure of a nucleic acid ligand that binds thrombinin Padmanabhan, K. et al., J. Biol. Chem. 24, 17651 (1993); Wang, K. Y.et al., Biochemistry 32, 1899 (1993); and Macaya, R. F. et al., Proc.Nat'l. Acad. Sci. USA 90, 3745 (1993). Selected aptamers may beresynthesized using one or more modified bases, sugars or backbonelinkages. Aptamers consist essentially of the minimum sequence ofnucleic acid needed to confer binding specificity, but may be extendedon the 5′ end, the 3′ end, or both, or may be otherwise derivatized orconjugated. ps Small Molecules

[0121] The term “small molecule” includes any chemical or other moiety,other than polypeptides and nucleic acids, that can act to affectbiological processes. Small molecules can include any number oftherapeutic agents presently known and used, or can be small moleculessynthesized in a library of such molecules for the purpose of screeningfor biological function(s). Small molecules are distinguished frommacromolecules by size. The small molecules of this invention usuallyhave molecular weight less than about 5,000 daltons (Da), preferablyless than about 2,500 Da, more preferably less than 1,000 Da, mostpreferably less than about 500 Da.

[0122] Small molecules include without limitation organic compounds,peptidomimetics and conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other thanmacromolecules such nucleic acids and polypeptides. In addition tocarbon, organic compounds may contain calcium, chlorine, fluorine,copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and otherelements. An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocylcic compounds,imidizoles and phenols. An organic compound as used herein also includesnitrated organic compounds and halogenated (e.g., chlorinated) organiccompounds. Methods for preparing peptidomimetics are described below.Collections of small molecules, and small molecules identified accordingto the invention are characterized by techniques such as acceleratormass spectrometry (AMS; see Turteltaub et al., Curr Pharm Des 20006:991-1007, Bioanalytical applications of accelerator mass spectrometryfor pharmaceutical research; and Enjalbal et al., Mass Spectrom Rev 200019:139-61, Mass spectrometry in combinatorial chemistry.)

[0123] Preferred small molecules are relatively easier and lessexpensively manufactured, formulated or otherwise prepared. Preferredsmall molecules are stable under a variety of storage conditions.Preferred small molecules may be placed in tight association withmacromolecules to form molecules that are biologically active and thathave improved pharmaceutical properties. Improved pharmaceuticalproperties include changes in circulation time, distribution,metabolism, modification, excretion, secretion, elimination, andstability that are favorable to the desired biological activity.Improved pharmaceutical properties include changes in the toxicologicaland efficacy characteristics of the chemical entity.

[0124] Peptidomimetics

[0125] In general, a polypeptide mimetic (“peptidomimetic”) is amolecule that mimics the biological activity of a polypeptide, but thatis not peptidic in chemical nature. While, in certain embodiments, apeptidomimetic is a molecule that contains no peptide bonds (that is,amide bonds between amino acids), the term peptidomimetic may includemolecules that are not completely peptidic in character, such aspseudo-peptides, semi-peptides and peptoids. Examples of somepeptidomimetics by the broader definition (e.g., where part of apolypeptide is replaced by a structure lacking peptide bonds) aredescribed below. Whether completely or partially non-peptide incharacter, peptidomimetics according to this invention may provide aspatial arrangement of reactive chemical moieties that closely resemblesthe three-dimensional arrangement of active groups in a polypeptide. Asa result of this similar active-site geometry, the peptidomimetic mayexhibit biological effects that are similar to the biological activityof a polypeptide.

[0126] There are several potential advantages for using a mimetic of agiven polypeptide rather than the polypeptide itself. For example,polypeptides may exhibit two undesirable attributes, i.e., poorbioavailability and short duration of action. Peptidomimetics are oftensmall enough to be both orally active and to have a long duration ofaction. There are also problems associated with stability, storage andimmunoreactivity for polypeptides that may be obviated withpeptidomimetics.

[0127] Candidate, lead and other polypeptides having a desiredbiological activity can be used in the development of peptidomimeticswith similar biological activities. Techniques of developingpeptidomimetics from polypeptides are known. Peptide bonds can bereplaced by non-peptide bonds that allow the peptidomimetic to adopt asimilar structure, and therefore biological activity, to the originalpolypeptide. Further modifications can also be made by replacingchemical groups of the amino acids with other chemical groups of similarstructure, shape or reactivity. The development of peptidomimetics canbe aided by determining the tertiary structure of the originalpolypeptide, either free or bound to a ligand, by NMR spectroscopy,crystallography and/or computer-aided molecular modeling. Thesetechniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal polypeptide (Dean (1994), BioEssays, 16: 683-687; Cohen andShatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993),Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15:124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993),Sci. Am., 269: 92-98, all incorporated herein by reference].

[0128] Specific examples of peptidomimetics are set forth below. Theseexamples are illustrative and not limiting in terms of the other oradditional modifications.

[0129] Peptides With A Reduced Isostere Pseudopeptide Bond

[0130] Proteases act on peptide bonds. Substitution of peptide bonds bypseudopeptide bonds may confer resistance to proteolysis or otherwisemake a compound less labile. A number of pseudopeptide bonds have beendescribed that in general do not affect polypeptide structure andbiological activity. The reduced isostere pseudopeptide bond is asuitable pseudopeptide bond that is known to enhance stability toenzymatic cleavage with no or little loss of biological activity(Couder, et al. (1993), Int. J. Polypeptide Protein Res. 41:181-184,incorporated herein by reference). Thus, the amino acid sequences ofthese compounds may be identical to the sequences of their parentL-amino acid polypeptides, except that one or more of the peptide bondsare replaced by an isostere pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution wouldconfer resistance to proteolysis by exopeptidases acting on theN-terminus.

[0131] Peptides With A Retro-Inverso Pseudopeptide Bond

[0132] To confer resistance to proteolysis, peptide bonds may also besubstituted by retro-inverso pseudopeptide bonds (Dalpozzo, et al.(1993), Int. J. Polypeptide Protein Res. 41:561-566, incorporated hereinby reference). According to this modification, the amino acid sequencesof the compounds may be identical to the sequences of their L-amino acidparent polypeptides, except that one or more of the peptide bonds arereplaced by a retro-inverso pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution willconfer resistance to proteolysis by exopeptidases acting on theN-terminus.

[0133] Peptoid Derivatives

[0134] Peptoid derivatives of polypeptides represent another form ofmodified polypeptides that retain the important structural determinantsfor biological activity, yet eliminate the peptide bonds, therebyconferring resistance to proteolysis (Simon, et al., 1992, Proc. Natl.Acad. Sci. USA, 89:9367-9371 and incorporated herein by reference).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid.

[0135] Polypeptides

[0136] The polypeptides of this invention, including the analogs andother modified variants, may generally be prepared following knowntechniques. Preferably, synthetic production of the polypeptide of theinvention may be according to the solid phase synthetic method. Forexample, the solid phase synthesis is well understood and is a commonmethod for preparation of polypeptides, as are a variety ofmodifications of that technique [Merrifield (1964), J. Am. Chem. Soc.,85: 2149; Stewart and Young (1984), Solid Phase polypeptide Synthesis,Pierce Chemical Company, Rockford, Ill.; Bodansky and Bodanszky (1984),The Practice of polypeptide Synthesis, Springer-Verlag, New York;Atherton and Sheppard (1989), Solid Phase polypeptide Synthesis: APractical Approach, IRL Press, New York].

[0137] Alternatively, polypeptides of this invention may be prepared inrecombinant systems using polynucleotide sequences encoding thepolypeptides. For example, fusion proteins are typically prepared usingrecombinant DNA technology.

[0138] Polypeptide Derivatives

[0139] A “derivative” of a polypeptide is a compound that is not, bydefinition, a polypeptide, i.e., it contains at least one chemicallinkage that is not a peptide bond. Thus, polypeptide derivativesinclude without limitation proteins that naturally undergopost-translational modifications such as, e.g., glycosylation. It isunderstood that a polypeptide of the invention may contain more than oneof the following modifications within the same polypeptide. Preferredpolypeptide derivatives retain a desirable attribute, which may bebiological activity; more preferably, a polypeptide derivative isenhanced with regard to one or more desirable attributes, or has one ormore desirable attributes not found in the parent polypeptide.

[0140] Mutant Polypeptides: A polypeptide having an amino acid sequenceidentical to that found in a protein prepared from a natural source is a“wildtype” polypeptide. Mutant oligopeptides can be prepared by chemicalsynthesis, including without limitation combinatorial synthesis.

[0141] Mutant polypeptides larger than oligopeptides can be preparedusing recombinant DNA technology by altering the nucleotide sequence ofa nucleic acid encoding a polypeptide. Although some alterations in thenucleotide sequence will not alter the amino acid sequence of thepolypeptide encoded thereby (“silent” mutations), many will result in apolypeptide having an altered amino acid sequence that is alteredrelative to the parent sequence. Such altered amino acid sequences maycomprise substitutions, deletions and additions of amino acids, with theproviso that such amino acids are naturally occurring amino acids.

[0142] Thus, subjecting a nucleic acid that encodes a polypeptide tomutagenesis is one technique that can be used to prepare mutantpolypeptides, particularly ones having substitutions of amino acids butno deletions or insertions thereof. A variety of mutagenic techniquesare known that can be used in vitro or in vivo including withoutlimitation chemical mutagenesis and PCR-mediated mutagenesis. Suchmutagenesis may be randomly targeted (i.e., mutations may occur anywherewithin the nucleic acid) or directed to a section of the nucleic acidthat encodes a stretch of amino acids of particular interest. Using suchtechniques, it is possible to prepare randomized, combinatorial orfocused compound libraries, pools and mixtures.

[0143] Polypeptides having deletions or insertions of naturallyoccurring amino acids may be synthetic oligopeptides that result fromthe chemical synthesis of amino acid sequences that are based on theamino acid sequence of a parent polypeptide but which have one or moreamino acids inserted or deleted relative to the sequence of the parentpolypeptide. Insertions and deletions of amino acid residues inpolypeptides having longer amino acid sequences may be prepared bydirected mutagenesis.

[0144] Chemically Modified Polypeptides: As contemplated by thisinvention, the term “polypeptide” includes those having one or morechemical modification relative to another polypeptide, i.e., chemicallymodified polypeptides. The polypeptide from which a chemically modifiedpolypeptide is derived may be a wildtype protein, a mutant protein or amutant polypeptide, or polypeptide fragments thereof; an antibody orother polypeptide ligand according to the invention including withoutlimitation single-chain antibodies, bacterial proteins and polypeptidederivatives thereof; or polypeptide ligands prepared according to thedisclosure. Preferably, the chemical modification(s) confer(s) orimprove(s) desirable attributes of the polypeptide but does notsubstantially alter or compromise the biological activity thereof.Desirable attributes include but are limited to increased shelf-life;enhanced serum or other in vivo stability; resistance to proteases; andthe like. Such modifications include by way of non-limiting exampleN-terminal acetylation, glycosylation, and biotinylation.

[0145] Polypeptides with N-Terminal or C-Terminal Chemical Groups: Aneffective approach to confer resistance to peptidases acting on theN-terminal or C-terminal residues of a polypeptide is to add chemicalgroups at the polypeptide termini, such that the modified polypeptide isno longer a substrate for the peptidase. One such chemical modificationis glycosylation of the polypeptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of polypeptides in human serum(Powell et al. (1993), Pharma. Res. 10: 1268-1273). Other chemicalmodifications which enhance serum stability include, but are not limitedto, the addition of an N-terminal alkyl group, consisting of a loweralkyl of from 1 to 20 carbons, such as an acetyl group, and/or theaddition of a C-terminal amide or substituted amide group.

[0146] Polypeptides with a Terminal D-Amino Acid: The presence of anN-terminal D-amino acid increases the serum stability of a polypeptidethat otherwise contains L-amino acids, because exopeptidases acting onthe N-terminal residue cannot utilize a D-amino acid as a substrate.Similarly, the presence of a C-terminal D-amino acid also stabilizes apolypeptide, because serum exopeptidases acting on the C-terminalresidue cannot utilize a D-amino acid as a substrate. With the exceptionof these terminal modifications, the amino acid sequences ofpolypeptides with N-terminal and/or C-terminal D-amino acids are usuallyidentical to the sequences of the parent L-amino acid polypeptide.

[0147] Polypeptides With Substitution of Natural Amino Acids ByUnnatural Amino Acids: Substitution of unnatural amino acids for naturalamino acids in a subsequence of a polypeptide can confer or enhancedesirable attributes including biological activity. Such a substitutioncan, for example, confer resistance to proteolysis by exopeptidasesacting on the N-terminus. The synthesis of polypeptides with unnaturalamino acids is routine and known in the art (see, for example, Coller,et al. (1993), cited above).

[0148] Post-Translational Chemical Modifications: Different host cellswill contain different post-translational modification mechanisms thatmay provide particular types of post-translational modification of afusion protein if the amino acid sequences required for suchmodifications is present in the fusion protein. A large number (˜100) ofpost-translational modifications have been described, a few of which arediscussed herein. One skilled in the art will be able to chooseappropriate host cells, and design chimeric genes that encode proteinmembers comprising the amino acid sequence needed for a particular typeof modification.

[0149] Glycosylation is one type of post-translational chemicalmodification that occurs in many eukaryotic systems, and may influencethe activity, stability, pharmacogenetics, immunogenicity and/orantigenicity of proteins. However, specific amino acids must be presentat such sites to recruit the appropriate glycosylation machinery, andnot all host cells have the appropriate molecular machinery.Saccharomyces cerevisieae and Pichia pastoris provide for the productionof glycosylated proteins, as do expression systems that utilize insectcells, although the pattern of glyscoylation may vary depending on whichhost cells are used to produce the fusion protein.

[0150] Another type of post-translation modification is thephosphorylation of a free hydroxyl group of the side chain of one ormore Ser, Thr or Tyr residues. Protein kinases catalyze such reactions.Phosphorylation is often reversible due to the action of a proteinphosphatase, an enzyme that catalyzes the dephosphorylation of aminoacid residues.

[0151] Differences in the chemical structure of amino terminal residuesresult from different host cells, each of which may have a differentchemical version of the methionine residue encoded by a start codon, andthese will result in amino termini with different chemicalmodifications.

[0152] For example, many or most bacterial proteins are synthesized withan amino terminal amino acid that is a modified form of methionine, i.e,N-formyl-methionine (fMet). Although the statement is often made thatall bacterial proteins are synthesized with an fMet initiator aminoacid; although this may be true for E. coli, recent studies have shownthat it is not true in the case of other bacteria such as Pseudomonasaeruginosa (Newton et al., J. Biol. Chem. 274:22143-22146, 1999). In anyevent, in E. coli, the formyl group of fMet is usually enzymaticallyremoved after translation to yield an amino terminal methionine residue,although the entire fMet residue is sometimes removed (see Hershey,Chapter 40, “Protein Synthesis” in: Escherichia Coli and SalmonellaTyphimurium: Cellular and Molecular Biology, Neidhardt, Frederick C.,Editor in Chief, American Society for Microbiology, Washington, D.C.,1987, Volume 1, pages 613-647, and references cited therein.) E. colimutants that lack the enzymes (such as, e.g., formylase) that catalyzesuch post-translational modifications will produce proteins having anamino terminal fMet residue (Guillon et al., J. Bacteriol.174:4294-4301, 1992).

[0153] In eukaryotes, acetylation of the initiator methionine residue,or the penultimate residue if the initiator methionine has been removed,typically occurs co- or post-translationally. The acetylation reactionsare catalyzed by N-terminal acetyltransferases (NATs, a.k.a.N-alpha-acetyltransferases), whereas removal of the initiator methionineresidue is catalyzed by methionine aminopeptidases (for reviews, seeBradshaw et al., Trends Biochem. Sci. 23:263-267, 1998; and Driessen etal., CRC Crit. Rev. Biochem. 18:281-325, 1985). Amino terminallyacetylated proteins are said to be “N-acetylated,” “N alpha acetylated”or simply “acetylated.” Another post-translational process that occursin eukaryotes is the alpha-amidation of the carboxy terminus. Forreviews, see Eipper et al. Annu. Rev. Physiol. 50:333-344, 1988, andBradbury et al. Lung Cancer 14:239-251, 1996. About 50% of knownendocrine and neuroendocrine peptide hormones are alpha-amidated(Treston et al., Cell Growth Differ. 4:911-920, 1993). In most cases,carboxy alpha-amidation is required to activate these peptide hormones.

[0154] Aminoglycosides

[0155] A class of small molecules of particular interest are known asaminoglycosides, particularly those that inhibit a sphingomyelinase(SMase), particularly a neutral SMase. Example 13 describes the use ofthe screening methods of the invention to prepare novel therapeuticagents using chemical libraries based on the aminoglycoside structure.

[0156] Aminoglycosides were first identified as antibiotics produced bymicroorganisms of the genus Micromonospora. The antibiotics, recoveredfrom the Micromonospora culture media, included Gentamicin (Weinstein etal., Antimicrobial Agents and Chemotherapy, 1963, page 1; Cooper et al.,J. Infect. Dis. 119:342, 1969; Waitz: Antimicrobial Agents andChemotherapy 2:464, 1972), Antibiotic No. 460 (Japanese Pat. No.16153/71), Sisomicin (Weinstein et al., J. Antibiotics 23:551, 555, 559,1970), Kanamycin, Neomycin, and many others as described below. For areview, see Edson et al., The Aminoglycosides, Mayo Clin Proc74:519-528, 1999.

[0157] Aminoglycosides are a group of antibiotics that exert theirbactericidal activity primarily by inhibition of protein synthesis.Aminoglycoside molecules bind to the bacterial 30S ribosomal subunitrendering the ribosomes unavailable for translation, which results incell death.

[0158] The first aminoglycoside, streptomycin, was isolated fromStreptomyces griseus in 1943. Neomycin, isolated from Streptomycesfradiae, had better activity than streptomycin against aerobicgram-negative bacilli but, because of its formidable toxicity, could notsafely be used systemically. Gentamicin, isolated from Micromonospora in1963, was a breakthrough in the treatment of gram-negative bacillaryinfections, including those caused by Pseudomonas aeruginosa. Otheraminoglycosides were subsequently developed, including amikacin(Amikin), netilmicin (Netromycin) and tobramycin (Nebcin), which are allcurrently available for systemic use in the United States.

[0159] Within the aminoglycoside family, the suffix “- mycin” is used inthe name when the antibiotic is produced by Streptomyces species and“micin,” when produced by Micromonospora species.

[0160] Structure of Aminoglycosides

[0161] Aminoglycosides are water soluble weak bases that are polycationsat body pH. They are chemically similar in that they have one of twobases to which is attached two or three aminosugars. The aminosugars arelinked to the by glycosidic bonds, hence the group name. The base instreptomycin is streptidine, but all of the others have2-deoxystreptamine so that most of the members differ in the number andnature of the aminosugars attached to the 2-deoxystreptamine.

[0162] The terms “aminoglycoside” is used herein according to itsconventional chemical meanings, as described in various standard textson organic chemistry. The following is a condensed summary of theseterms.

[0163] As implied by the term, an aminosaccharide is a saccharidemolecule (the term saccharide is used interchangeably with sugar) havingat least one amine group coupled to it, either directly or indirectly.Saccharide molecules (i.e., polyhydroxylated aldehydes or ketones) existas both straight chains and ring structures, which spontaneously convertback and forth between straight and ring forms in an equilibrium-type“tautomeric” mode. Since the equilibria between straight and ringstructures tends to generate more ring structures than straight chainsat any given moment when dissolved in an aqueous solvent, mostsaccharides are usually drawn and discussed as ring structures.

[0164] Saccharide rings are called furanose rings if the ring structureitself (excluding any pendant groups) contains five atoms, and pyranoserings if the ring contains six atoms. Most furanose molecules arederived from pentose sugars (i.e., sugars which contain five carbonatoms, such as ribose, arabinose, or xylose). In a pentose molecule, oneof the atoms in the ring form of the molecule is an oxygen atom; thefifth carbon atom is attached to the ring in a pendant structure,usually as a hydroxylated methyl group. In the same manner, mostpyranose molecules (with six-membered rings) are hexose sugars such asglucose, galactose, and mannose, or derivatives thereof. Hexose sugarscontain six carbon atoms; in the most common pyranose ring, five carbonsare in the ring along with an oxygen atom; the sixth carbon atom isattached to the ring in a pendant group.

[0165] A glycoside molecule contains at least one saccharide component(usually drawn as a ring) attached through an oxygen atom (which can beregarded as an ether linkage) to a second molecular group having atleast one carbon atom. If a glycoside molecule is chemically hydrolyzedto break the ether linkage(s), it will release at least one saccharidemolecule. Usually, the glycoside linkage is between adjacent sacchariderings, to form disaccharides, trisaccharides, polysaccharides, etc.

[0166] As implied by the name, an aminoglycoside is a glycoside with oneor more amino groups. Because of their biological properties,aminoglycosides are an important class of aminosaccharides. Neomycin A(neamine), Neomycin B and C, Gentamicin, sisomycin, streptomycin, andtobramycin are all aminoglycosides, since they have the requisite aminegroups, saccharide rings, and oxygen linkages. Most aminoglycosides wereinitially identified due to the anti-bacterial activities of variousmicrobes that synthesize such compounds in nature. Many of theseaminoglycoside antibiotics can be altered or derivatized in various waysthat do not destroy their antibiotic activity; for example, if NeomycinB or Neomycin C is cleaved between the disaccharide structure and thepentose ring, the two cleavage products are Neomycin A (a disaccharide,also known as neamine) and either Neobiosamine B or Neobiosamine C.

[0167] The aminoglycosides and derivatives thereof that are ofparticular interest to the present invention have the structure:

[0168] Wherein each of R1-R13 is independently hydrogen, alkyl,optionally substituted alkyl, alkenyl, optionally substituted alkenyl,alkynyl, optionally substituted alkynyl, aryl, optionally substitutedaryl, cycloalkyl, optionally substituted cycloalkyl, alkoxy, optionallysubstituted alkoxy, heterocyclic, optionally substituted heterocyclic,heteroaryl, optionally substituted heteroaryl, hydroxyl, halogen, nitro,carboxyl, thioalkyl, amino, alkylamino, arylamino, amido, ammonium,alkylammonium, sulfonyl, aminosulfonyl, alkylsulfonyl, alkoxycarbonyl,acetyl, or acyl.

[0169] In one embodiment, each of R1-R13 is independently hydrogen,alkyl, optionally substituted alkyl, alkoxy, optionally substitutedalkoxy, cycloalkyl, optionally substituted cycloalkyl, cyclooxyalkyl,optionally substituted cyclooxyalkyl, hydroxyl, halogen, amino,alkylamino, amido, ammonium, alkoxycarbonyl, acetyl, or acyl.

[0170] In another embodiment, each of R1-R13 is independently hydrogen,alkyl, optionally substituted alkyl, hydroxyl, alkoxy, optionallysubstituted alkoxy, halogen, amino, acetyl, or acyl. In a relatedembodiment, at least one of R1-R13 is independently acetyl.

[0171] In another embodiment, at least one of R2, R3, R6, R12, or R13 ishalogen. In a related embodiment, the halogen is fluorine.

[0172] In another embodiment, at at least one of R4, R5, or R6 is acyl.

[0173] In another embodiment, R7 is alkyl or optionally substitutedalkyl. In a related embodiment, the optionally substituted alkyl is a C6to C12 alkyl.

[0174] In another embodiment, each of R1-R13 is independently hydrogen,alkyl, optionally substituted alkyl, alkenyl, optionally substitutedalkenyl, alkynyl, optionally substituted alkynyl, alkoxy, optionallysubstituted alkoxy, aryl, optionally substituted aryl, cycloalkyl,optionally substituted cycloalkyl, heterocyclic, optionally substitutedheterocyclic, heteroaryl, optionally substituted heteroaryl, hydroxyl,halogen, nitro, carboxyl, thioalkyl, amino, alkylamino, arylamino,amido, ammonium, alkylammonium, sulfonyl, aminosulfonyl, alkylsulfonyl,alkoxycarbonyl, acetyl, or acyl, with the proviso that when R6=H, R7=H,R8=CH3, R9=OH, R10=CH3, R11=H, R12=OH, and R13=OH; if R1=NH2, R2=H,R3=H, and R4=CH3, then R5 is not NH2 or NHCH3; and if R1=OH, R2=OH,R3=OH, and R4 is H, then R5 is not NH2.

[0175] In another embodiment, R1=NH2, R2=H, R3=H, R4=CH3, R5=NH2 orNHCH3, R6=H, R7=H, R8=CH3, R9=OH, R10=CH3, R11=H, R12=OH, and R13=OH.

[0176] In another embodiment, R1=OH, R2=OH, R3=OH, R4=H, R5=NH2, R6=H,R7=H, R8=CH3, R9=OH, R10=CH3, R11=H, R12=OH, and R13=OH.

[0177] In addition to the above, some compounds are also calledaminoglycosides even though they do not have a glycosidic oxygenlinkage, since they are components of larger molecules which are trueaminoglycosides, and they are commonly synthesized using bacterialaminoglycosides as starting reagents. Examples include2,6-diamino-2,6-dideoxy-D-glucose (which can be obtained by hydrolyzingNeomycin) and streptidine (which can be obtained by hydrolyzingstreptomycin). As used herein, the term “aminoglycoside” encompassesthese compounds as well.

[0178] Gentamicin and derivatives thereof are one type of aminoglycosideof interest, as gentamicin is known to inhibit SMase (Ghosh et al., J.Biol. Chem. 262:12550-12556, 1987). Several isoforms of gentamicin areknown, including Gentamicin C, Gentamicin C1a, Gentamicin C2, GentamicinC26 and Gentamicin B (see Example 13 for the structures of theseisoforms). Methods of preparing gentamicin isoforms and derivatives aredisclosed in U.S. Pat. Nos. 3,984,395 (Method of isolating gentamicinC2a); 4,288,547 (Fermentative process for preparing antibiotics of thegentamicin class); and 5,814,488 (Semisynthetic 1-N-ethylgentamicin C1aand method for its preparation). Gentamicin derivatives are disclosed inU.S. Pat. Nos. 4,387,219 (2-Hydroxy gentamicin compounds); 4,283,528(1-N-aminohydroxyacyl derivatives of gentamicin B); and 4,223,024(4″-O-Alkylgentamicins and sagamicins). U.S. Pat. No. 4,150,949,(Immunoassay for gentamicin), discloses fluorescein-labelledgentamicins, including fluoresceinthiocarbanyl gentamicin.

[0179] Kanamycin and derivatives thereof are of interest. Manychemically modified derivatives of kanamycin are known; for a review,see Mingeot-Leclercq et al., Antimicrobial Agents and Chemoctherapy43:727-737, 1999. Kanamycin derivatives are described in U.S. Pat. Nos.4,873,225 (1-n-(4-amino-3-fluoro-2-hydroxybutyryl)-kanamycins);4,455,419 (2′-Modified kanamycins and production thereof); 4,424,343(Preparation of 1-N->.omega.-amino-. alpha.-hydroxyalkanoyl!kanamycinpolysilylates and products); 4,337,336 (Derivative of kanamycin A and aprocess for the preparation thereof); 4,195,170 (3′,4′-Episulfidokanamycin B compounds); 4,178,437 (1-N-Kanamycin derivatives); 4,170,642(Derivatives of kanamycin A); 4,140,849 (Kanamycin C derivatives);4,120,955 (Method for production of kanamycin C and its derivatives);3,974,137 (Process for the preparation of1-[L-(−)-.gamma.-amino-.alpha.-hydroxybutyryl]-kanamycin A (RD-1341A));3,940,382 (1,2′-Di-N-substituted kanamycin B compounds); 4,178,437(1-N-Kanamycin derivatives); 4,140,849 (Kanamycin C derivatives);4,181,797 (1-N-(.omega.-amino-.alpha.-hydroxyalkanoyl) derivatives of4′-deoxy-6′-N-methylkanamycin A); and 4,051,315 (6″-Deoxykanamycin B and6″-deoxytobramycin).

[0180] Fortimicin and derivatives thereof are of interest. Fortimicin isa naturally occurring aminoglycoside antibiotic, first produced byfermentation of a microorganism belonging to the genus Micromonospora.Studies of fortimicin showed that blocking the 2-hydroxy group byinactivates the antibiotic. As a result, much attention was focused ondeveloping chemical modifications to the aminoglycoside that position inorder to develop more stable fortimicin derivatives. Fortimicin andchemical derivatives of Fortimicin are described in U.S. Pat. Nos.4,214,079 (4-N, 2′-N and 4,2′-Di-N-fortimicin AL derivatives); 4,214,078(Fortimicin AL); 4,214,076 (2′-N-Substituted fortimicin B andderivatives); 4,220,756 (Method of producing 3-O-demethylfortimicinB,4-N-alkylfortimicin B derivatives and related aminoglycosideantibiotics); 4,219,644 (Fortimicins AH and AI); 4,219,643 (FortimicinAN); 4,219,642 (Fortimicin AO); 4,214,080 (Fortimicins AM and AP);4,214,075 (6′-Epi-fortimicin A and B derivatives); 4,213,974 (4-N,2′-Nand 4,2′-Di-N-fortimicin AO derivatives); 4,213,972 (4-N, 2′-N and4,2′Di-N-fortimicins AH and Al); 4,213,971 (4-N, 2′-N and4,2′-Di-N-fortimicin AD derivatives); 4,207,415 (Method of producing2-deoxyfortimicin A); 4,205,070 (6′N-Alkyl- and 6′,6′-di-N-alkylderivatives of fortimicins A and B); 4,176,178 (2-Deoxy-2′-N-acyl andalkyl fortimicins A and B); 4,169,198 (2-Deoxyfortimicin B); 4,183,920(4-N-Acyl, 2′-N-acyl and 4,2′-N,N′-diacylfortimicin E derivatives);4,192,867 (2-Deoxyfortimicin A, 4-N-alkyl and 4-N-acyl-2-deoxyfortimicinB derivatives); 4,220,756 (Method of producing 3-O-demethylfortimicinB,4-N-alkylfortimicin B derivatives and related aminoglycosideantibiotics); 4,196,197 (2′N-Acyl and alkyl-6′-N-alkyl- and6′,6′-di-N-alkyl derivatives of fortimicins A and B); 4,187,299(Fortimicin E); 4,187,298 (2′N-acyl and alkyl fortimicin B andderivatives, 4,2′-N,N′diacyl and dialkyl fortimicin B derivatives4-N-acyl-2′-N-alkyl and 4-N-alkyl-2′-N-acyl fortimicin B derivatives);4,187,297 (3-De-O-methyl-2-N-acyl and alkyl fortimicins A and B);4,187,296 (2-N-acyl and alkyl 6-epi-fortimicin B and derivatives);4,208,407 (5-Deoxyfortimicin A, 2,5-dideoxyfortimicin A and thecorresponding 4-N-acyl and alkyl fortimicin B derivatives thereof andintermediates therefor); 4,216,210 (Fortimicins AM and AP derivatives);4,319,022 (2-O-Substituted sulfonyl derivatives of fortimicin B);4,251,516 (2-Deoxy-3-O-Demethylfortimicins); 4,251,511 (Antibiotic andfermentation process of preparing); 4,250,304 (2-Deoxy-2-substitutedfortimicin A and B and derivatives); 4,317,904 (1,2-EpiminofortimicinB); 4,255,421 (Fortimicin aminoglycosides, process for productionthereof, and use thereof); 4,252,972 (FortimicinB-1,2:4,5-bis-carbamates); 4,418,193 (Method of producing2-epi-fortimicin A); and 4,207,415 (Method of producing2-deoxyfortimicin A).

[0181] Sisomicin and derivatives thereof are disclosed in U.S. Pat. Nos.4,438,260 (Sisomicin compounds); 4,369,251 (Method for the production ofsisomicin); 4,365,020 (Method for the preparation of antibioticsisomicin); 4,336,369 (Selectively protected1-N-(.omega.-aminoalkoxycarbonyl)-sisomicin derivatives); 4,312,859(Sisomicin derivatives, processes for their production and theirmedicinal use); 3,997,524 (Process for the manufacture of 6′-N-alkylderivatives of sisomicin and verdamicin; novel intermediates usefultherein, and novel 6′-N-alkylverdamicins prepared thereby); and3,988,316 (Antibiotics sisomicin and verdamicin I and complex containingsame).

[0182] Amikacin and derivatives thereof are disclosed in U.S. Pat. Nos.5,763,587 (Process for the synthesis of amikacin); 5,656,735 (Processfor the preparation of amikacin precursors); 5,621,085 (Process for thepreparation of amikacin precursors); 4,985,549 (Process for preparingamikacin); and 4,902,790 (Novel process for the synthesis of amikacin).

[0183] Dibekacin and derivatives thereof are disclosed in U.S. Pat. Nos.5,618,795 (Dibekacin derivatives and arbekacin derivatives activeagainst resistant bacteria, and the production thereof); and 5,488,038(Dibekacin derivatives and arbekacin derivatives active againstresistant bacteria).

[0184] Other aminoglycosides are disclosed in U.S. Pat. Nos. 4,855,287(Aminoglycoside compounds, processes for production thereof, andpharamaceutical composition containing the same); 5,442,047 (Process forpreparing isepamicin); 4,208,531 (Synthetic aminoglycosides); 4,656,160(Aminoglycoside derivatives); 4,647,656 (Aminoglycoside compounds);4,645,760 (Activated aminoglycosides and aminoglycoside-aminocyclitolspharmaceutical compositions and method of use); 4,617,293 (Flavonoidphosphate salts of aminoglycoside antibiotics); 4,554,269 (Kasugamycinderivatives, pharmaceutical compositions and method of use); 4,503,046(1-Nitro-aminoglycoside derivatives, pharmaceutical compositionscontaining them and such derivatives for use as pharmaceuticals);4,493,831 (Aminoglycoside derivatives); 4,486,418 (2′-Deaminoaminoglycosides and composition thereof); 4,468,513 (2′-N-Acylated and2′-N-alkylated derivatives of 4-O-substituted-2-deoxystreptamineaminoglycosides); 4,468,512 (1-N-Acylated and 1-N-alkylated derivativesof 4-O-substituted-2-deoxystreptamine aminoglycosides); 4,438,107(Aminoglycosides and use thereof); 4,424,345 (1-N-Acylated and1-N-alkylated derivatives of 4-O-substituted-2-deoxystreptamineaminoglycosides and process); 4,424,344 (2-N-Acylated and 2-N-alkylatedderivatives of 4-O-substituted-2-deoxystreptamine aminoglycosides andprocess); 4,380,625 (Process for the preparation of purifiedaminoglycoside antibiotics); 4,349,667 (Aminoglycoside antibioticG-367-2); 4,347,354 (Preparation of1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl]aminoglycoside polysilylatedantibiotics and products obtained therefrom); 4,330,673 (Process forproducing 3-O-demethylaminoglycoside and novel 3-O-demethylfortimicinderivatives); 4,297,486 (Aminoglycoside antibiotic G-367-1 and methodfor the production thereof); 4,297,485 (Production of a selectivelyprotected N-acylated derivative of an aminoglycosidic antibiotic);4,279,997 (Process for production of aminoglycoside antibiotics);4,273,923 (Process for preparing aminoglycoside derivatives); 4,255,421(Fortimicin aminoglycosides, process for production thereof, and usethereof); 4,252,972 (Fortimicin B-1,2:4,5-bis-carbamates); 4,250,170(Antibacterial agents Bu-2349A and B and method of using same);4,248,865 (Novel aminoglycoside derivatives); 4,242,331 (Aminoglycosidesand method of use); 4,230,847 (Aminoglycoside antibiotic compounds);4,226,978 (beta.-Galactosyl-umbelliferone-labeled aminoglycosideantibiotics and intermediates in their preparation); 4,223,022Stabilized aminoglycoside antibiotic formulations); 4,217,446(.omega.Amino-2-hydroxyalkyl derivatives of aminoglycoside antibiotics);4,214,074 Hydroxyalkyl derivatives of aminoglycoside antibiotics);4,212,859(2′-Hydroxy-2′-desamino-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols,methods for their manufacture, method for their use as antibacterialagents, and compositions useful therefor: 4,209,511 (Aminoglycosideantibiotics and process for production thereof); 4,207,314(Isofortimicin); 4,201,774 (Novel aminoglycoside derivatives); 4,200,628(Novel aminoglycoside derivatives); 4,199,570 1-N-Hetero containingaminoglycoside derivatives); 4,189,569 (Seldomycin factor 5derivatives); 4,187,372 (Seldomycin factor 5 derivative); 4,187,299(Fortimicin E); 4,170,643 (Aminoglycoside-aminocyclitol derivatives andmethod of use); 4,166,114 (Aminoglycoside antibiotic derivatives andmethod of use); 4,146,617 (Desoxystreptamine derivatives, salts,pharmaceutical compositions and method of use); 4,136,254 (Process ofselectively blocking amino functions in aminoglycosides using transitionmetal salts and intermediates used thereby); 4,125,707 (Protectedpseudotrisaccharide intermediate for paromomycin and neomycinderivatives); 4,117,221 (Aminoacyl derivatives of aminoglycosideantibiotics); 4,107,435 (Process for .omega.-amino-2-hydroxyalkylderivatives of aminoglycoside antibiotics); 4,101,556 (Total synthesisof 2,5-dideoxystreptamines); 4,085,208 (Process for preparing4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols and novel 1-epimers and1-N-alkyl derivatives produced thereby; methods for the use of the1-epimer derivatives as antibacterial agents and compositions usefultherefor); 4,066,752 1-Desamino-1-hydroxy and1-desamino-1-epi-hydroxy-4,6-di-o-(aminoglycosyl)-1,3-diaminocyclitols;1 -desamino-1-oxo-4,6-di-o-(aminoglycosyl)-1,3-diaminocyclitols,intermediates and use as antibacterial agents); 4,065,615(Deoxyaminoglycoside antibiotic derivatives); 4,064,339 (Antibioticaminoglycosides, processes of preparation and pharmaceuticalcompositions); 4,049,498 (Methods for the preparation of semi-syntheticaminocyclitol aminoglycoside antibiotics); 4,044,123(6′-N-alkyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols, methods fortheir use as antibacterial agents and compositions useful therefor);4,038,478 (O-Glycoside ortho esters of neamine containing compounds);4,032,404 (Fermentation process for producing apramycin and nebramycinfactor V′); 4,031,210 (Antibiotic aminoglycosides, processes ofpreparation and pharmaceutical compositions); 4,024,332 (Aminoglycosideantibiotics and intermediates therefor); 4,020,269(Epiminodeaminodeoxyaminoglycoside antibiotics and intermediates);4,012,576 (Antibiotic complex Bu 2183); 4,011,390 (Semi-syntheticaminocyclitol aminoglycoside antibiotics and methods for the preparationthereof); 4,009,328 (Aminoglycoside 66-40C, method for its manufacture,method for its use as an intermediate in the preparation of knownantibiotics and novel antibacterials); 4,003,922 (Synthesis ofcis-1,4-cyclohexadiene dioxide); 4,002,608(1-N-alkyl-aminoglycoside-XK-88 derivatives and methods for theirmanufacture); 3,996,205 (Aminoglycoside antibiotics and intermediates);3,984,393 (Aminoglycoside antibiotics); 3,981,861 (Antibioticaminoglycosides, processes of preparation and pharmaceuticalcompositions); 3,978,214 (Novel4,6-di-O-(aminoglycosyl)-2-deoxystreptamine, method for its manufacture,method for its use as an antiprotozoal agent and compositions usefulthereof); 3,962,429 (Method for reducing side effects of aminoglycosideantibiotics and composition therefor); 3,959,255 (Antibioticaminoglycosides, and process of preparation); 3,953,422 (Deoxyglucosederivatives); and 3,953,293 (Process for the preparation of xylostasin).

[0185] Additional aminoglycosides are disclosed by Matsumoto et al.,“Synthesis of novel 13-methyl-13-dihydroanthracyclines”, Chem Pharm Bull(Tokyo), 34:4613-9, 1986; Israel et al., “Adriamycin analogues.Preparation and biological evaluation of someN-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamycin derivatives”, JMed Chem., 2:1273-6, 1986; Takahashi et al., “Production of novelantibiotic, dopsisamine, by a new subspecies of Nocardiopsis mutabiliswith multiple antibiotic resistance”, J Antibiot (Tokyo), 39:175-83,1986; Yasuda et al., “Total synthesis of 3-O-demethylsporaricin A”, JAntibiot (Tokyo), 38:1512-25, 1985; Tsunakawa et al., “Inosamycin, acomplex of new aminoglycoside antibiotics. I. Production, isolation andproperties”, J Antibiot (Tokyo), 38:1302-12, 1985; Matsuhashi et al.,“In vitro and in vivo antibacterial activities of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with those of otheraminoglycoside antibiotics”, Antimicrob Agents Chemother, 27:589-94,1985; Matsunaga et al., “Bacterial uptake of habekacin, a novelaminoglycoside antibiotic”, J Antibiot (Tokyo), 37:596-601, 1984; Tanakaet al., “Mechanism of action of habekacin, a novel amino acid-containingaminoglycoside antibiotic”, Antimicrob Agents Chemother, 24:797-802,1983; Dawson, “Activity of SC33428, a novel bishydrazone-bridgedderivative of 4-demethoxydaunorubicin, against experimental tumors inmice”, Cancer Res., 43:2880-3, 1983; Digranes et al., “N-formimidoylthienamycin: in vitro comparison with cefoxitin and tobramycin againstclinical, bacterial isolates”, Acta Pathol Microbiol Immunol Scand [B],91:141-4, 1983; FitzGerald et al., “3,4-Dihydroxybenzylamine: animproved dopamine analog cytotoxic for melanoma cells in part throughoxidation products inhibitory to dna polymerase”, J Invest Dermatol.,80:119-23, 1983; Israel et al., “Adriamycin analogues. Novel anomericribofuranoside analogues of daunorubicin”, J Med Chem., 25:28-31, 1982;Tanaka, “Effects of habekacin, a novel aminoglycoside antibiotic, onexperimental corneal ulceration due to Pseudomonas aeruginosa”, JAntibiot (Tokyo), 34:892-7, 19881; Fujiwara et al., “Production of a newaminoglycoside antibiotic by a mutant of Bacillus circulans”, J Antibiot(Tokyo), 33:836-41, 1980; Ohashi et al., “In vitro and in vivoantibacterial activity of KW1070, a new aminoglycoside antibiotic”,Antimicrob Agents Chemother., 17:138-43, 1980; Inouye et al., “A novelaminoglycoside antibiotic, substance SF-2052”, J Antibiot (Tokyo),32:1355-6, 1979; Perzynski et al., “Effects of apramycin, a novelaminoglycoside antibiotic on bacterial protein synthesis”, Eur JBiochem., 99:623-8, 1979; Suzuki et al., “Preparation and somemicrobiological properties of novel kanamycin-glucoside derivatives”, JAntibiot (Tokyo), 32:753-5, 1979; Smith et al., “Synthesis ofdaunorubicin analogues with novel 9-acyl substituents”, J Med Chem.,22:40-4, 1979; Davies et al., “Semisynthetic aminoglycosideantibacterials. 6. Synthesis of sisomicin, Antibiotic G-52, and novel6′-substituted analogues of sisomicin from aminoglycoside 66-40C”, J MedChem., 21:189-93, 1978; Egan et al., “Fortimicins A and B, newaminoglycoside antibiotics. III. Structural identification”, J Antibiot(Tokyo), 30:552-63, 1977; Okachi et al., “Fortimicins A and B, newaminoglycoside antibiotics. II. Isolation, physico-chemical andchromatographic properties”, J Antibiot (Tokyo), 30:541-51, 1977;Kinumaki et al., “Macrolide antibiotics M-4365 produced byMicromonospora. II. Chemical structures”, J Antibiot (Tokyo), 30:450-4,1977; Okutani et al., “Conversion of aminoglycosidic antibiotics: Noveland efficient approaches to 3′-deoxyaminoglycosides via 3′-phosphorylesters”, J Am Chem Soc., 99:1278-9, 1977; Hanessian et al.,“Aminoglycoside antibiotics: oxidative degradations leading to novelbiochemical probes and synthetic intermediates”, J Antibiot (Tokyo),28:835-7, 1975; Reimann et al., “The structure of sisomicin, a novelunsaturated aminocyclitol antibiotic from Micromonospora inyoensis”, JOrg Chem., 39:1451-7, 1974; Kugelman et al., “Letter: The preparation ofgaramine, a novel pseudodisaccharide from sisomycin”, J Antibiot(Tokyo), 26:394-5, 1973; Arcamone et al., “Adriamycin(14-hydroxydaunomycin), a novel antitumor antibiotic”, TetrahedronLett., 13:1007-10, 1969; Matsumoto et al., “Synthesis of novel13-methyl-13-dihydroanthracyclines”, Chem Pharm Bull (Tokyo), 34:4613-9,1986; Israel et al., “Adriamycin analogues. Preparation and biologicalevaluation of someN-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamycin derivatives”, JMed Chem., 29:1273-6, 1986; Takahashi et al., “Production of novelantibiotic, dopsisamine, by a new subspecies of Nocardiopsis mutabiliswith multiple antibiotic resistance”, J Antibiot (Tokyo), 39:175-83,1986; Yasuda et al., “Total synthesis of 3-O-demethylsporaricin A”, JAntibiot (Tokyo), 38:1512-25, 1985; Tsunakawa et al., “Inosamycin, acomplex of new aminoglycoside antibiotics. I. Production, isolation andproperties”, J Antibiot (Tokyo), 38:1302-12, 1985; Matsuhashi et al.,“In vitro and in vivo antibacterial activities of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with those of otheraminoglycoside antibiotics”, Antimicrob Agents Chemother., 27:589-94,1985; Matsunaga et a 1., “Bacterial uptake of habekacin, a novelaminoglycoside antibiotic”, J Antibiot (Tokyo), 37:596-601, 1984; Tanakaet al., “Mechanism of action of habekacin, a novel amino acid-containingaminoglycoside antibiotic”, Antimicrob Agents Chemother., 24:797-802,1983; Dawson, “Activity of SC33428, a novel bishydrazone-bridgedderivative of 4-demethoxydaunorubicin, against experimental tumors inmice”, Cancer Res., 43:2880-3, 1983; Digranes et al., “N-formimidoylthienamycin: in vitro comparison with cefoxitin and tobramycin againstclinical, bacterial isolates”, Acta Pathol Microbiol Immunol Scand [B].,91:141-4, 1983; FitzGerald et al., “3,4-Dihydroxybenzylamine: animproved dopamine analog cytotoxic for melanoma cells in part throughoxidation products inhibitory to dna polymerase”, J Invest Dermatol.,80:119-23, 1983; Israel et al., “Adriamycin analogues. Novel anomericribofuranoside analogues of daunorubicin”, J Med Chem., 25:28-31, 1982;Tanaka, “Effects of habekacin, a novel aminoglycoside antibiotic, onexperimental corneal ulceration due to Pseudomonas aeruginosa”, JAntibiot (Tokyo), 34:892-7, 1981; Fujiwara et al., “Production of a newaminoglycoside antibiotic by a mutant of Bacillus circulans”, J Antibiot(Tokyo), 33:836-41, 1980; Ohashi et al., “In vitro and in vivoantibacterial activity of KW1070, a new aminoglycoside antibiotic”,Antimicrob Agents Chemother., 17:138-43, 1980; Inouye et al., “A novelaminoglycoside antibiotic, substance SF-2052”, J Antibiot (Tokyo),32:1355-6, 1979; Perzynski et al., “Effects of apramycin, a novelaminoglycoside antibiotic on bacterial protein synthesis”, Eur JBiochem., 99:623-8, 1979; Suzuki et al., “Preparation and somemicrobiological properties of novel kanamycin-glucoside derivatives”, JAntibiot (Tokyo), 32:753-5, 1979; Smith et al., “Synthesis ofdaunorubicin analogues with novel 9-acyl substituents”, J Med Chem.,22:40-4, 1979; Davies et al., “Semisynthetic aminoglycosideantibacterials. 6. Synthesis of sisomicin, Antibiotic G-52, and novel6′-substituted analogues of sisomicin from aminoglycoside 66-40C”, J MedChem., 21:189-93, 1978; Egan et al., “Fortimicins A and B, newaminoglycoside antibiotics. III. Structural identification”, J Antibiot(Tokyo), 30:552-63, 1977; Okachi et al., “Fortimicins A and B, newaminoglycoside antibiotics. II. Isolation, physico-chemical andchromatographic properties”, J Antibiot (Tokyo), 30:541-51, 1977;Kinumaki et al., “Macrolide antibiotics M-4365 produced byMicromonospora. II. Chemical structures”, J Antibiot (Tokyo), 30:450-4,1977; Okutani et al., “Conversion of aminoglycosidic antibiotics: Noveland efficient approaches to 3′-deoxyaminoglycosides via 3′-phosphorylesters”, J Am Chem Soc., 99:1278-9, 1977; Hanessian et a 1.,“Aminoglycoside antibiotics: oxidative degradations leading to novelbiochemical probes and synthetic intermediates”, J Antibiot (Tokyo),28:835-7, 1975; Reimann et al., “The structure of sisomicin, a novelunsaturated aminocyclitol antibiotic from Micromonospora inyoensis”, JOrg Chem., 39:1451-7, 1974; Kugelman et al., “Letter: The preparation ofgaramine, a novel pseudodisaccharide from sisomycin”, J Antibiot(Tokyo), 26:394-5, 1973; Arcamone et al., “Adriamycin(14-hydroxydaunomycin), a novel antitumor antibiotic”, TetrahedronLett., 13:1007-10, 1969; Yew et al., “New antimycobacterial agents”,Monaldi Arch Chest Dis., 51:394-404, 1996; Urban et al., “Comparativein-vitro activity of SCH 27899, a novel everninomicin, and vancomycin”,J Antimicrob Chemother, 37:361-4, 1996; Lam et al., “Production andisolation of two novel esperamicins in a chemically defined medium”, JAntibiot (Tokyo), 48:1497-501, 1995; Pelyvas et al., “Novelaminocyclitol antibiotics derived from natural carbohydrates”, CarbohydrRes., 272:C5-9, 1995; Pelyvas et al., “Synthesis of newpseudodisaccharide aminoglycoside antibiotics from carbohydrates”, JAntibiot (Tokyo), 48:683-95, 1995; Jones, “Isepamicin (SCH 21420,1-N-HAPA gentamicin B): microbiological characteristics includingantimicrobial potency of spectrum of activity”, J Chemother., 2:7-16,1995 Suppl; Abe et al., “Novel antitumor antibiotics, saptomycins. II.Isolation, physico-chemical properties and structure elucidation”, JAntibiot (Tokyo), 46:1536-49, 1993; Abe et al., “Novel antitumorantibiotics, saptomycins. I. Taxonomy of the producing organism,fermentation, HPLC analysis and biological activities”, J Antibiot(Tokyo), 46:1530-5, 1993; Phillipson et al., “Lanomycin andglucolanomycin, antifungal agents produced by Pycnidiophora dispersa.II. Structure elucidation”, J Antibiot (Tokyo), 45:313-9, 1992; Marianiet al., “In vitro activity of novel sulphonic derivatives of distamycinA”, EXS., 61:455-8, 1992; Abe et al., “Novel antitumor antibiotics,saptomycins D and E”, J Antibiot (Tokyo), 44:908-11, 1991; Priebe etal., “3′-Hydroxyesorubicin. Synthesis and antitumor activity”, JAntibiot (Tokyo), 43:838-46, 1990; Brill et al., “Altromycins, novelpluramycin-like antibiotics. II. Isolation and elucidation ofstructure”, J Antibiot (Tokyo), 43:229-37, 1990; Jackson et al.,“Altromycins, novel pluramycin-like antibiotics. I. Taxonomy of theproducing organism, fermentation and antibacterial activity”, J Antibiot(Tokyo), 43(3):223-8, 1990; Flynn et al., “The chiral synthesis andbiochemical properties of electron rich phenolic sulfoxide analogs ofsparsomycin”, Biochem Biophys Res Commun., 166:673-80, 1990; Kitamura etal., “Pirarubicin, a novel derivative of doxorubicin. THP-COP therapyfor non-Hodgkin's lymphoma in the elderly”, Am J. Clin Oncol., 13 Suppl1:S15-9, 1990; Gu et al., “In vitro activity of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with activities of otheraminoglycosides”, Antimicrob Agents Chemother, 33:1998-2003, 1989; Lam,“Biosynthesis of elsamicin A, a novel antitumor antibiotic”, J. NatProd., 52:1015-21, 1989; Rolston et al., “In vitro activity oftrospectomycin (U-63366F), a novel spectinomycin analog, againstgram-positive isolates from cancer patients”, Eur J Clin MicrobiolInfect Dis., 8:254-60, 1989; Gupta et al., “Synthesis, cytotoxicity, andantiviral activity of some acyclic analogues of thepyrrolo[2,3-d]pyrimidine nucleoside antibiotics tubercidin, toyocamycin,and sangivamycin”, J Med Chem., 32:402-8, 1989; Hochlowski et al.,“Phenelfamycins, a novel complex of elfamycin-type antibiotics. II.Isolation and structure determination” J Antibiot (Tokyo), 41:1300-15,1988; Jackson et al., “Phenelfamycins, a novel complex of elfamycin-typeantibiotics. I. Discovery, taxonomy and fermentation”, J Antibiot(Tokyo), 41:1293-9, 1988; Saitoh et al., “Boholmycin, anewaminoglycoside antibiotic. I. Production, isolation and properties”, JAntibiot (Tokyo), 41:855-61, 1988; Matsumoto et al., “Synthesis of novel13-methyl-13-dihydroanthracyclines”, Chem Pharm Bull (Tokyo), 34:4613-9,1986; Israel et al., “Adriamycin analogues. Preparation and biologicalevaluation of someN-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamycin derivatives”, JMed Chem., 29:1273-6, 1986; Takahashi et al., “Production of novelantibiotic, dopsisamine, by a new subspecies of Nocardiopsis mutabiliswith multiple antibiotic resistance”, J Antibiot (Tokyo), 39:175-83,1986; Yasuda et al., “Total synthesis of 3-0-demethylsporaricin A”, JAntibiot (Tokyo), 38:1512-25, 1985; Tsunakawa et al., “Inosamycin, acomplex of new aminoglycoside antibiotics. I. Production, isolation andproperties”, J Antibiot (Tokyo), 38:1302-12, 1985; Matsuhashi et al.,“In vitro and in vivo antibacterial activities of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with those of otheraminoglycoside antibiotics”, Antimicrob Agents Chemother., 27:589-94,1985; Matsunaga et al., “Bacterial uptake of habekacin, a novelaminoglycoside antibiotic”, J Antibiot (Tokyo), 37:596-601, 1984; Tanakaet al., “Mechanism of action of habekacin, a novel amino acid-containingaminoglycoside antibiotic”, Antimicrob Agents Chemother., 24:797-802,1983; Dawson, “Activity of SC33428, a novel bishydrazone-bridgedderivative of 4-demethoxydaunorubicin, against experimental tumors inmice”, Cancer Res., 43:2880-3, 1983; Digranes et al., “N-formimidoylthienamycin: in vitro comparison with cefoxitin and tobramycin againstclinical, bacterial isolates”, Acta Pathol Microbiol Immunol Scand [B],91:141-4, 1983; FitzGerald et al., “3,4-Dihydroxybenzylamine: animproved dopamine analog cytotoxic for melanoma cells in part throughoxidation products inhibitory to dna polymerase”, J Invest Dermatol.,80:119-23, 1983; Israel et al., “Adriamycin analogues. Novel anomericribofuranoside analogues of daunorubicin”, J Med Chem., 25:28-31, 1982;Tanaka, “Effects of habekacin, a novel aminoglycoside antibiotic, onexperimental corneal ulceration due to Pseudomonas aeruginosa”, JAntibiot (Tokyo), 34:892-7, 1981; Fujiwara et al., “Production of a newaminoglycoside antibiotic by a mutant of Bacillus circulans”, J Antibiot(Tokyo), 33:836-41, 1980; Ohashi et al., “In vitro and in vivoantibacterial activity of KW1070, a new aminoglycoside antibiotic”,Antimicrob Agents Chemother., 17:138-43, 1980; Inouye et al., “A novelaminoglycoside antibiotic, substance SF-2052”, J Antibiot (Tokyo),32:1355-6, 1979; Perzynski et al., “Effects of apramycin, a novelaminoglycoside antibiotic on bacterial protein synthesis”, Eur JBiochem., 99:623-8, 1979; Suzuki et al., “Preparation and somemicrobiological properties of novel kanamycin-glucoside derivatives”, JAntibiot (Tokyo), 32:753-5, 1979; Smith et al., “Synthesis ofdaunorubicin analogues with novel 9-acyl substituents”, J Med Chem.,22:40-4, 1979; Davies et al., “Semisynthetic aminoglycosideantibacterials. 6. Synthesis of sisomicin, Antibiotic G-52, and novel6′-substituted analogues of sisomicin from aminoglycoside 66-40C”, J MedChem., 21:189-93, 1978; Egan et al., “Fortimicins A and B, newaminoglycoside antibiotics. III. Structural identification”, J Antibiot(Tokyo), 30:552-63, 1977; Okachi et al., “Fortimicins A and B, newaminoglycoside antibiotics. II. Isolation, physico-chemical andchromatographic properties”, J Antibiot (Tokyo), 30:541-51, 1977;Kinumaki et al., “Macrolide antibiotics M-4365 produced byMicromonospora. II. Chemical structures”, J Antibiot (Tokyo), 30:450-4,1977; Okutani et al., “Conversion of aminoglycosidic antibiotics: Noveland efficient approaches to 3′-deoxyaminoglycosides via 3′-phosphorylesters”, J Am Chem Soc., 99:1278-9, 1977; Hanessian et al.,“Aminoglycoside antibiotics: oxidative degradations leading to novelbiochemical probes and synthetic intermediates”, J Antibiot (Tokyo),28:835-7, 1975; Reimann et al., “The structure of sisomicin, a novelunsaturated aminocyclitol antibiotic from Micromonospora inyoensis”, JOrg Chem., 39:1451-7, 1974; Kugelman et al., “Letter: The preparation ofgaramine, a novel pseudodisaccharide from sisomycin”, J Antibiot(Tokyo), 26:394-5, 1973; Arcamone et al., “Adriamycin(14-hydroxydaunomycin), a novel antitumor antibiotic”, TetrahedronLett., 13:1007-10, 1969; Yew et al., “New antimycobacterial agents”,Monaldi Arch Chest Dis., 51:394-404, 1996; Urban et al., “Comparativein-vitro activity of SCH 27899, a novel everninomicin, and vancomycin”,J Antimicrob Chemother., 37:361-4, 1996; Lam et al., “Production andisolation of two novel esperamicins in a chemically defined medium”, JAntibiot (Tokyo), 48:1497-501, 1995; Pelyvas et al., “Novelaminocyclitol antibiotics derived from natural carbohydrates”, CarbohydrRes., 272:C5-9, 1995; Pelyvas et al., “Synthesis of newpseudodisaccharide aminoglycoside antibiotics from carbohydrates”, JAntibiot (Tokyo), 48:683-95, 1995; Jones, “Isepamicin (SCH 21420,1-N-HAPA gentamicin B): microbiological characteristics includingantimicrobial potency of spectrum of activity”, J Chemother., 7 Suppl2:7-16, 1995; Abe et al., “Novel antitumor antibiotics, saptomycins. II.Isolation, physico-chemical properties and structure elucidation”, JAntibiot (Tokyo), 46:1536-49, 1993; Abe et al., “Novel antitumorantibiotics, saptomycins. I. Taxonomy of the producing organism,fermentation, HPLC analysis and biological activities”, J Antibiot(Tokyo), 46:1530-5, 1993; Phillipson et al., “Lanomycin andglucolanomycin, antifungal agents produced by Pycnidiophora dispersa.II. Structure elucidation”, J Antibiot (Tokyo), 45:313-9, 1992; Marianiet al., “In vitro activity of novel sulphonic derivatives of distamycinA”, EXS., 61:455-8, 1992; Abe et al., “Novel antitumor antibiotics,saptomycins D and E”, J Antibiot (Tokyo), 44:908-11, 1989; Priebe etal., “3′-Hydroxyesorubicin. Synthesis and antitumor activity”, JAntibiot (Tokyo), 43:838-46, 1990; Brill et al., “Altromycins, novelpluramycin-like antibiotics. II. Isolation and elucidation ofstructure”, J Antibiot (Tokyo), 43:229-37, 1990; Jackson et al.,“Altromycins, novel pluramycin-like antibiotics. I. Taxonomy of theproducing organism, fermentation and antibacterial activity”, J Antibiot(Tokyo), 43:223-8, 1990; Flynn et al., “The chiral synthesis andbiochemical properties of electron rich phenolic sulfoxide analogs ofsparsomycin”, Biochem Biophys Res Commun., 166:673-80, 1990; Kitamura etal., “Pirarubicin, a novel derivative of doxorubicin. THP-COP therapyfor non-Hodgkin's lymphoma in the elderly”, Am J Clin Oncol., 13 Suppl1:S 15-9, 1990; Gu et al., “In vitro activity of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with activities of otheraminoglycosides”, Antimicrob Agents Chemother., 33:1998-2003, 1989; Lamet al., “Biosynthesis of elsamicin A, a novel antitumor antibiotic”, JNat Prod. 52:1015-21, 1989; Rolston et al., “In vitro activity oftrospectomycin (U-63366F), a novel spectinomycin analog, againstgram-positive isolates from cancer patients”, Eur J Clin MicrobiolInfect Dis., 8:254-60, 1989; Gupta et al., “Synthesis, cytotoxicity, andantiviral activity of some acyclic analogues of thepyrrolo[2,3-d]pyrimidine nucleoside antibiotics tubercidin, toyocamycin,and sangivamycin”, J Med Chem., 32:402-8, 1989; Hochlowski et al.,“Phenelfamycins, a novel complex of elfamycin-type antibiotics. II.Isolation and structure determination”, J Antibiot (Tokyo), 41:1300-15,1988; Jackson et al., “Phenelfamycins, a novel complex of elfamycin-typeantibiotics. I. Discovery, taxonomy and fermentation”, J Antibiot(Tokyo), 41(10):1293-9, 1988; Saitoh et al., “Boholmycin, anewaminoglycoside antibiotic. I. Production, isolation and properties”, JAntibiot (Tokyo), 41:855-61, 1988; Yew et al., “New antimycobacterialagents”, Monaldi Arch Chest Dis., 51(5):394-404, 1996; Urban et al.,“Comparative in-vitro activity of SCH 27899, a novel everninomicin, andvancomycin”, J Antimicrob Chemother, 37:361-4, 1996; Lam et al.,“Production and isolation of two novel esperamicins in a chemicallydefined medium”, J Antibiot (Tokyo), 148:1497-501, 1995; Pelyvas et al.,“Novel aminocyclitol antibiotics derived from natural carbohydrates”,Carbohydr Res., 272:C5-9, 1995; Plyvas et al., “Synthesis of newpseudodisaccharide aminoglycoside antibiotics from carbohydrates”, JAntibiot (Tokyo), 48:683-95, 1995; Jones, “Isepamicin (SCH 21420,1-N-HAPA gentamicin B): microbiological characteristics includingantimicrobial potency of spectrum of activity”, J Chemother., 7 Suppl2:7-16, 1995; Abe et al., “Novel antitumor antibiotics, saptomycins. II.Isolation, physico-chemical properties and structure elucidation”, JAntibiot (Tokyo), 46:1536-49, 1993; Abe et al., “Novel antitumorantibiotics, saptomycins. I. Taxonomy of the producing organism,fermentation, HPLC analysis and biological activities”, J Antibiot(Tokyo), 46:1530-5, 1993; Phillipson et al., “Lanomycin andglucolanomycin, antifungal agents produced by Pycnidiophora dispersa.II. Structure elucidation”, J Antibiot (Tokyo), 45:313-9, 1992; Marianiet al., “In vitro activity of novel sulphonic derivatives of distamycinA”, EXS., 61:455-8, 1992; Abe et al., “Novel antitumor antibiotics,saptomycins D and E”, J Antibiot (Tokyo), 44:908-11, 1991; Priebe etal., “3′-Hydroxyesorubicin. Synthesis and antitumor activity”, JAntibiot (Tokyo), 43:838-46, 1990; Brill et al., “Altromycins, novelpluramycin-like antibiotics. II. Isolation and elucidation ofstructure”, J Antibiot (Tokyo), 43:229-37, 1990; Jackson et al.,“Altromycins, novel pluramycin-like antibiotics. I. Taxonomy of theproducing organism, fermentation and antibacterial activity”, J Antibiot(Tokyo), 43:223-8, 1990; Flynn et al., “The chiral synthesis andbiochemical properties of electron rich phenolic sulfoxide analogs ofsparsomycin”, Biochem Biophys Res Commun., 166:673-80, 1990; Kitamura etal., “Pirarubicin, a novel derivative of doxorubicin. THP-COP therapyfor non-Hodgkin's lymphoma in the elderly”, Am J Clin Oncol., 13 Suppl1:S15-9, 1990; Gu et al., “In vitro activity of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with activities of otheraminoglycosides”, Antimicrob Agents Chemother., 33:1998-2003, 1989; Lamet al., “Biosynthesis of elsamicin A, a novel antitumor antibiotic”, JNat Prod., 52:1015-21, 1989; Rolston et al., “In vitro activity oftrospectomycin (U-63366F), a novel spectinomycin analog, againstgram-positive isolates from cancer patients”, Eur J Clin MicrobiolInfect Dis., 8:254-60, 1989; Gupta et al., “Synthesis, cytotoxicity, andantiviral activity of some acyclic analogues of thepyrrolo[2,3-d]pyrimidine nucleoside antibiotics tubercidin, toyocamycin,and sangivamycin”, J Med Chem., 32:402-8, 1989; Hochlowski et al.,“Phenelfamycins, a novel complex of elfamycin-type antibiotics. II.Isolation and structure determination”, J Antibiot (Tokyo), 41:1300-15,1988; Jackson et al., “Phenelfamycins, a novel complex of elfamycin-typeantibiotics. I. Discovery, taxonomy and fermentation”, J Antibiot(Tokyo), 41:1293-9, 1988; Saitoh et al., “Boholmycin, a newaminoglycoside antibiotic. I. Production, isolation and properties”, JAntibiot (Tokyo), 41:855-61, 1988; Miller et al., “Clinical pharmacologyand toxicity of 4′-O-tetrahydropyranyladriamycin”, Cancer Res.,47:1461-5, 1987; Stefani et al., “First microbiological approach todactimicin, a novel aminoglycoside antibiotic”, Drugs Exp Clin Res.,13:727-9, 1987; Matsumoto et al., “Synthesis of novel13-methyl-13-dihydroanthracyclines”, Chem Pharm Bull (Tokyo), 34:4613-9,1986; Israel et al., “Adriamycin analogues. Preparation and biologicalevaluation of someN-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamycin derivatives”, JMed Chem., 29:1273-6, 1986; Takahashi et al., “Production of novelantibiotic, dopsisamine, by a new subspecies of Nocardiopsis mutabiliswith multiple antibiotic resistance”, J Antibiot (Tokyo), 39:175-83,1986; Yasuda et al., “Total synthesis of 3-O-demethylsporaricin A”, JAntibiot (Tokyo), 38:1512-25, 1985; Tsunakawa et al., “Inosamycin, acomplex of new aminoglycoside antibiotics. I. Production, isolation andproperties”, J Antibiot (Tokyo), 38:1302-12, 1985; Matsuhashi et al.,“In vitro and in vivo antibacterial activities of dactimicin, a novelpseudodisaccharide aminoglycoside, compared with those of otheraminoglycoside antibiotics”, Antimicrob Agents Chemother., 27:589-94,1985; Matsunaga et al., “Bacterial uptake of habekacin, a novelaminoglycoside antibiotic”, J Antibiot (Tokyo), 37:596-601, 1984; Tanakaet al., “Mechanism of action of habekacin, a novel amino acid-containingaminoglycoside antibiotic”, Antimicrob Agents Chemother., 24:797-802,1983; Dawson, “Activity of SC33428, a novel bishydrazone-bridgedderivative of 4-demethoxydaunorubicin, against experimental tumors inmice”, Cancer Res., 43:2880-3, 1983; Digranes et al., “N-formimidoylthienamycin: in vitro comparison with cefoxitin and tobramycin againstclinical, bacterial isolates”, Acta Pathol Microbiol Immunol Scand [B],91:141-4, 1983; FitzGerald et al., “3,4-Dihydroxybenzylamine: animproved dopamine analog cytotoxic for melanoma cells in part throughoxidation products inhibitory to dna polymerase”, J Invest Dermatol.,80:119-23, 1983; Israel et al., “Adriamycin analogues. Novel anomericribofuranoside analogues of daunorubicin”, J Med Chem.e 25:28-31, 1982;Tanaka. “Effects of habekacin, a novel aminoglycoside antibiotic, onexperimental corneal ulceration due to Pseudomonas aeruginosa”, JAntibiot (Tokyo), 34:892-7, 1981; Fujiwara et al., “Production of a newaminoglycoside antibiotic by a mutant of Bacillus circulans”, J Antibiot(Tokyo), 33:836-41, 1980; Ohashi et al., “In vitro and in vivoantibacterial activity of KW1070, a new aminoglycoside antibiotic”,Antimicrob Agents Chemother, 17:138-43, 1980; Inouye et al., “A novelaminoglycoside antibiotic, substance SF-2052”, J Antibiot (Tokyo),32:1355-6, 1979; Perzynski et al., “Effects of apramycin, a novelaminoglycoside antibiotic on bacterial protein synthesis”, Eur JBiochem, 99:623-8, 1979; Suzuki et al., “Preparation and somemicrobiological properties of novel kanamycin-glucoside derivatives”, JAntibiot (Tokyo), 32:753-5, 1979; Smith et al., “Synthesis ofdaunorubicin analogues with novel 9-acyl substituents”, J Med Chem.,22:40-4, 1979; Davies et al., “Semisynthetic aminoglycosideantibacterials. 6. Synthesis of sisomicin, Antibiotic G-52, and novel6′-substituted analogues of sisomicin from aminoglycoside 66-40C”, J MedChem., 21:189-93, 1978; Egan et al., “Fortimicins A and B, newaminoglycoside antibiotics. III. Structural identification”, J Antibiot(Tokyo), 30:552-63, 1977; Okachi et al., “Fortimicins A and B, newaminoglycoside antibiotics. II. Isolation, physico-chemical andchromatographic properties”, J Antibiot (Tokyo), 30:541-51, 1977;Kinumaki et al., “Macrolide antibiotics M-4365 produced byMicromonospora. II. Chemical structures”, J Antibiot (Tokyo), 30:450-4.1977; Okutani et al., “Conversion of aminoglycosidic antibiotics: Noveland efficient approaches to 3′-deoxyaminoglycosides via 3′-phosphorylesters”, J Am Chem Soc., 99(4):1278-9, 1977; Hanessian et al.,“Aminoglycoside antibiotics: oxidative degradations leading to novelbiochemical probes and synthetic intermediates”, J Antibiot (Tokyo),28:835-7, 1975; Reimann et al., “The structure of sisomicin, a novelunsaturated aminocyclitol antibiotic from Micromonospora inyoensis”, JOrg Chem., 39:1451-7, 1974; Kugelman et al., “Letter: The preparation ofgaramine, a novel pseudodisaccharide from sisomycin”, J Antibiot(Tokyo), 26:394-5, 1973; Arcamone et al., “Adriamycin(14-hydroxydaunomycin), a novel antitumor antibiotic”, TetrahedronLett., 13:1007-10, 1969; Marchini et al.,“4-Demethoxy-3′-deamino-3′-aziridinyl-4′-methylsulphonyl-daunorubicin(PNU-159548), a novel anticancer agent active against tumor cell lineswith different resistance mechanisms”, Cancer Res., 61:1991-5, 2001;Sunada et al., “Acetylation of aminoglycoside antibiotics with6′-methylamino group, istamycin B and micronomicin, by a novelaminoglycoside 6′-acetyltransferase of actinomycete origin”, J Antibiot(Tokyo), 53:1416-9, 2000; Ganguly, “Ziracin, a novel oligosaccharideantibiotic”, J Antibiot (Tokyo), 53:1038-44, 2000; Nicolaou et al.,“Total synthesis of everninomicin 13,384-1—Part 2: synthesis of theFGHA2 fragment”, Chemistry, 6:3116-48, 2000; Nicolaou et al., “Totalsynthesis of everninomicin 13,384-1—Part 1: retrosynthetic analysis andsynthesis of the A1B(A)C fragment”, Chemistry, 6:3095-115, 2000; Wang etal., “In vivo activity and pharmacokinetics of ziracin (SCH27899), a newlong-acting everninomicin antibiotic, in a murine model ofpenicillin-susceptible or penicillin-resistant pneumococcal pneumonia”,Antimicrob Agents Chemother., 44:1010-8, 2000; Ganguly et al., “Chemicalmodifications and structure activity studies of ziracin and relatedeverninomicin antibiotics”, Bioorg Med Chem Lett., 9:1209-14, 1999; andHotta et al., “The novel enzymatic 3″-N-acetylation of arbekacin by anaminoglycoside 3-N-acetyltransferase of Streptomyces origin and theresulting activity”, J Antibiot (Tokyo), 51:735-42, 1998.

[0186] Aminoglycosides Pharmacology

[0187] After injection, aminoglycosides are distributed mainly in theECF. Protein binding is low. Even with inflammation, concentrations intissues and secretions are much less than those in plasma levels.

[0188] Aminoglycosides are excreted unchanged into the urine byglomerular filtration. They generally have the same half-life in plasmaof 2 to 3 h; with renal insufficiency and in the elderly, the half-liferises markedly. To avoid toxicity, the maintenance dosages ofaminoglycosides in patients with renal insufficiency must be modified byeither decreasing the dose or increasing the interval between doses orboth.

[0189] Because of the distribution properties of aminoglycosides, dosingin obese patients should be based on a weight equal to lean body weightplus 50% of the adipose mass. In patients with excessive ECF, as inedema, the dose should be calculated based on total body weight.Patients with bums and cystic fibrosis have decreased plasma levels andmay require higher doses. Anemia tends to increase plasma levels.

[0190] One large aminoglycoside dose given once daily rather thanseveral divided doses given on multiple occasions throughout the day isbelieved to result in less net transfer of aminoglycoside from the bloodinto the tissue. This is believed to be accomplished by saturating therate by which aminoglycoside is moved into the tissue.

[0191] Treatment of patients with certain aminoglycosides results in theundesirable side effects of nephrotoxity and ototoxicity. (For a review,see Mingeot-Leclerq et al., Antimicrobial Agents and Chemotherapy43:1003-1012, 1999). The compounds used in the methods of the inventionare preferably not toxic, in particular because they would not typicallybe given for prolonged periods of time. Compounds identified by thescreening assays of the invention are tested for their toxicity inanimal models in order to identify nontoxic compounds. Aminoglycosideshaving undesirable side-effects can be administered to a patient withone or more agents that ameriolate or prevent the undesirable sideeffects. Additionally or alternatively, in the methods of the invention,the compounds are administered using a dosage regimen that is designedto minimize or avoid toxicity or any other undesirable side effects.

[0192] Modulation of the Sphingomyelin Signaling Pathway for TherapeuticBenefit

[0193] The sphingomyelin signaling pathway (a.k.a. the SM pathway or theceramide signaling pathway) is a “cascade” of biochemical events inwhich proteins in the pathway are activated (by enzymatic chemicalmodification or otherwise) with the end result that sphingosinemetabolism is affected. In most instances, activation of the SM pathwayleads to increased production of ceramide. For reviews of the molecularbiology of the sphingomyelin signaling pathway, see Hannun et al., Adv.Lipid Res. 25:27-41, 1993; Liu et al., Crit. Rev. Clin. Lab. Sci.36:511-573, 1999; Igarashi, J. Biochem. 122:1080-1087, 1997; and Oral etal., J. Biol. Chem. 272:4836-4842, 1997.

[0194] It has ben suggested that the sphingomyelin signal transductionpathway is activated during cardiac ischemia/hypoxia (Bielawska et al.,Am. J. Pathol. 151:1257-1263, 1997; Meldrum, Am. J. Physiol.274:R577-R595, 1998; and Cain et al., Mol. Cell. Cardiol. 31:931-947,1999). If so, one or more factors or processes may mediate theischemia-induced SPH production. One likely candidate for such amediator is the pro-inflammatory cytokine, tumor necrosis factor alpha(TNFα). In various animal models of ischemia, the myocardium producesTNFα (Squadrito et al., Eur. J. Pharmacol. 237:223-230, 1993; Herrmannet al., European Journal of Clinical Investigation 28:59-66, 1998;Meldrum et al., Ann. Thorac. Surg. 65:439-443, 1998). Recent evidencedemonstrates that the cardiomyocytes themselves produce TNFα and secretethe cytokine into the extracellular fluid (Comstock et al., J. Mol. CellCardiol. 30:2761-2775, 1998). Since TNFα receptors are expressed bycardiomyocytes (Krown et al., FEBS Letters 376:24-30, 1995; Torre-Amioneet al., Circulation 92:1487-1493, 1995), an autocrine/paracrine role forTNFα has been suggested (Meldrum et al., Ann. Thorac. Surg. 65:439-443,1998). Significantly, TNFα induces SPH production and apoptosis incardiac myocytes (Krown et al., J Clin. Invest. 98:2854-2865, 1996),presumably by acting by binding to the cardiomyocyte complement of TNFαreceptors Activation of the sphingomyelin signal transduction cascademay be a key early event in the cytotoxic (apoptotic) effects of thecytokine TNFα (Zhang et al., Endo. 136:4157-4160, 1995). TNFα can causesignificant apoptosis in cultured rat cardiomyocytes and it has beensuggested that TNFα-induced SPH production is responsible for the celldeath triggered by TNFα (Krown et al., J. Clin. Invest. 98:2854-2865,1996).

[0195] The SM pathway, many steps of which occur intracellularly, isinduced by a variety of extracellular stimuli. In sphingolipid-basedcardiovascular therapy, such stimuli may be inhibited or completelyblocked. SM pathway-inducing agents, the function of which may bemodulated, include but are not limited to cytokines. Cytokines ofparticular interest include but are not limited to pro-inflammatorycytokines, interferons and chemokines.

[0196] Methods of Screening for Novel Sphingolipid-Based TherapeuticAgents

[0197] The sphingolipid targets of the invention are readily adaptablefor use in high-throughput screening assays for screening candidatecompounds to identify those which have a desired activity, e.g.,inhibiting an enzyme that catalyzes a reaction that produces anundesirable sphingolipid, or blocking the binding of a sphingolipid to areceptor therefor. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as therapeuticagents.

[0198] The methods of screening of the invention comprise usingscreening assays to identify, from a library of diverse molecules, oneor more compounds having a desired activity. A “screening assay” is aselective assay designed to identify, isolate, and/or determine thestructure of, compounds within a collection that have a preselectedactivity. By “identifying” it is meant that a compound having adesirable activity is isolated, its chemical structure is determined(including without limitation determining the nucleotide and amino acidsequences of nucleic acids and polypeptides, respectively) the structureof and, additionally or alternatively, purifying compounds having thescreened activity). Biochemical and biological assays are designed totest for activity in a broad range of systems ranging fromprotein-protein interactions, enzyme catalysis, small molecule-proteinbinding, to cellular functions. Such assays include automated,semi-automated assays and HTS (high throughput screening) assays.

[0199] In HTS methods, many discrete compounds are preferably tested inparallel by robotic, automatic or semi-automatic methods so that largenumbers of test compounds are screened for a desired activitysimultaneously or nearly simultaneously. It is possible to assay andscreen up to about 6,000 to 20,000, and even up to about 100,000 to1,000,000 different compounds a day using the integrated systems of theinvention.

[0200] Typically in HTS, target molecules are contained in each well ofa multi-well microplate; in the case of enzymes, reactants are alsopresent in the wells. Currently, the most widely established techniquesutilize 96-well microtiter plates. In this format, 96 independent testsare performed simultaneously on a single 8 cm×12 cm plastic plate thatcontains 96 reaction wells. One or more blank wells contains all of thereactants except the candidate compound. Each of the non-standard wellscontain at least one candidate compound.

[0201] These wells typically require assay volumes that range from 50 to500 ul. In addition to the plates, many instruments, materials,pipettors, robotics, plate washers and plate readers are commerciallyavailable to fit the 96-well format to a wide range of homogeneous andheterogeneous assays. Microtiter plates with more wells, such as384-well microtiter plates, are also used, as are emerging methods suchas the nanowell method described by Schullek et al. (Anal Biochem., 30246, 20-29, 1997).

[0202] In one modality, screening comprises contacting a sphingolipidtarget with a diverse library of member compounds, some of which areligands of the target, under conditions where complexes between thetarget and ligands can form, and identifying which members of thelibraries are present in such complexes. In another non limitingmodality, screening comprises contacting a target enzyme with a diverselibrary of member compounds, some of which are inhibitors (oractivators) of the target, under conditions where a product or areactant of the reaction catalyzed by the enzyme produce a detectablesignal. In the latter modality, inhibitors of target enzyme decrease thesignal from a detectable product or increase a signal from a detectablereactant (or vice-versa for activators).

[0203] Chemical Libraries

[0204] Developments in combinatorial chemistry allow the rapid andeconomical synthesis of hundreds to thousands of discrete compounds.These compounds are typically arrayed in moderate-sized libraries ofsmall organic molecules designed for efficient screening. Combinatorialmethods, can be used to generate unbiased libraries suitable for theidentification of novel inhibitors. In addition, smaller, less diverselibraries can be generated that are descended from a single parentcompound with a previously determined biological activity. In eithercase, the lack of efficient screening systems to specifically targettherapeutically relevant biological molecules produced by combinationalchemistry such as inhibitors of important enzymes hampers the optimaluse of these resources.

[0205] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks,” such asreagents. For example, a linear combinatorial chemical library, such asa polypeptide library, is formed by combining a set of chemical buildingblocks (amino acids) in a large number of combinations, and potentaillyin every possible way, for a given compound length (i.e., the number ofamino acids in a polypeptide compound). Millions of chemical compoundscan be synthesized through such combinatorial mixing of chemicalbuilding blocks.

[0206] A “library” may comprise from 2 to 50,000,000 diverse membercompounds. Preferably, a library comprises at least 48 diversecompounds, preferably 96 or more diverse compounds, more preferably 384or more diverse compounds, more preferably, 10,000 or more diversecompounds, preferably more than 100,000 diverse members and mostpreferably more than 1,000,000 diverse member compounds. By “diverse” itis meant that greater than 50% of the compounds in a library havechemical structures that are not identical to any other member of thelibrary. Preferably, greater than 75% of the compounds in a library havechemical structures that are not identical to any other member of thecollection, more preferably greater than 90% and most preferably greaterthan about 99%.

[0207] The preparation of combinatorial chemical libraries is well knownto those of skill in the art. For reviews, see Thompson et al.,Synthesis and application of small molecule libraries, Chem Rev96:555-600, 1996; Kenan et al., Exploring molecular diversity withcombinatorial shape libraries, Trends Biochem Sci 19:57-64, 1994; Janda,Tagged versus untagged libraries: methods for the generation andscreening of combinatorial chemical libraries, Proc Natl Acad Sci USA.91:10779-85, 1994; Lebl et al., One-bead-one-structure combinatoriallibraries, Biopolymers 37:177-98, 1995; Eichler et al., Peptide,peptidomimetic, and organic synthetic combinatorial libraries, Med ResRev. 15:481-96, 1995; Chabala, Solid-phase combinatorial chemistry andnovel tagging methods for identifying leads, Curr Opin Biotechnol.6:632-9, 1995; Dolle, Discovery of enzyme inhibitors throughcombinatorial chemistry, Mol Divers. 2:223-36, 1997; Fauchere et al.,Peptide and nonpeptide lead discovery using robotically synthesizedsoluble libraries, Can J. Physiol Pharmacol. 75:683-9, 1997; Eichler etal., Generation and utilization of synthetic combinatorial libraries,Mol Med Today 1: 174-80, 1995; and Kay et al., Identification of enzymeinhibitors from phage-displayed combinatorial peptide libraries, CombChem High Throughput Screen 4:535-43, 2001.

[0208] Such combinatorial chemical libraries include, but are notlimited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175,Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton, et al.,Nature, 354:84-88 (1991)). Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to, peptoids (PCT Publication No. WO 91/19735); encodedpeptides (PCT Publication WO 93/20242); random bio-oligomers (PCTPublication No. WO 92/00091); benzodiazepines (U.S. Pat. No. 5,288,514);diversomers, such as hydantoins, benzodiazepines and dipeptides (Hobbs,et al., Proc. Nat. Acad. Sci. USA, 90:6909-6913 (1993)); vinylogouspolypeptides (Hagihara, et al., J. Amer. Chem. Soc. 114:6568 (1992));nonpeptidal peptidomimetics with .beta.-D-glucose scaffolding(Hirschmann, et al., J. Amer. Chem. Soc., 114:9217-9218 (1992));analogous organic syntheses of small compound libraries (Chen, et al.,J. Amer. Chem. Soc., 116:2661 (1994)); oligocarbamates (Cho, et al.,Science, 261:1303 (1993)); and/or peptidyl phosphonates (Campbell, etal., J. Org. Chem. 59:658 (1994)); nucleic acid libraries (see, Ausubel,Berger and Sambrook, all supra); peptide nucleic acid libraries (see,e.g., U.S. Pat. No. 5,539,083); antibody libraries (see, e.g., Vaughn,et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287);carbohydrate libraries (see, e.g., Liang, et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853); small organic molecule libraries(see, e.g., benzodiazepines, Baum C&E News, Jan. 18, page 33 (1993);isoprenoids (U.S. Pat. No. 5,569,588); thiazolidinones andmetathiazanones (U.S. Pat. No. 5,549,974); pyrrolidines (U.S. Pat. Nos.5,525,735 and 5,519,134); morpholino compounds (U.S. Pat. No.5,506,337); benzodiazepines (U.S. Pat. No. 5,288,514); and the like.

[0209] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem.Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Bio sciences, Columbia, Md., etc.).

[0210] Bioactive Lipid Libraries

[0211] Naturally occurring or synthetic lipids, particularlysphingolipids, are known that modulate processes in sphingolipidbiosynthesis and intracellular signaling by ceramide, S-1-P and othersphinglipids (for reviews of sphingolipid-mediated cell signalingprocesses and consequences arising therefrom, see Linn et al.,“Regulation of de novo sphingolipid biosynthesis and the toxicconsequences of its disruption”, Biochemical Society, 831-835, 2001;Luberto et al., “Sphingolipid Metabolism in the Regulation of BioactiveMolecules”, Lipids, 34:S5-S 11, 1999; Kester, “Sphingolipid Metabolitesand the Cellular Phenotype”, Trends in Glycoscience and Glycotechnology,9:447-460, 1997; Ariga et al., “Role of Sphingolipid-mediated cell deathin neurodegenerative diseases”, Journal of Lipid Research, 39:1-16,1998; Chan et al., “Ceramid Path in Human Lung Cell Death”, Am. J.Respir. Cell Mol. Biol., 22:460-468, 2000; and Hannun et al., “Ceramidein the eukaryotic stress response”, Cell Biology, 10:73-80, 2000; seealso Brownlee, Current Biology 11:R535-R538, 2001).

[0212] See, for example, Usta et al., “Structural Requirements ofCermaide and Sphingosine Based Inhibitors of Mitochondrial Ceramidase”,Biochemistry, 40:9657-9668, 2000; Hannun et al., “Method of InducingCellular Differentiations and Altering Cell Phenotype Using CeramideAnalogs”, U.S. Pat. No. 5,369,030, issued Nov. 29, 1994; Wei et al.,“Pharmaceutically Active Ceramide-Related Compounds”, U.S. Pat. No.5,631,394, issued May 20, 1997; Wei et al., “Methods of Treatment UsingPharmaceutically Active Ceramide-Related Compositions”, U.S. Pat. No.5,677,337, issued Oct. 14, 1997; Carson et al., “Compounds forInhibition of Ceramide-Mediated Signal Transduction”, U.S. Pat. No.6,323,201 B1, issued Nov. 27, 2001; Bell et al., “Inhibition of ProteinKinase C By Long-Chain Bases”, U.S. Pat. No. 4,937,232, issued Jun. 26,1990; Bell et al., “Inhibition of Protein Kinase C By Long-Chain Bases”,U.S. Pat. No. 4,816,450, issued Mar. 28, 1989; Hannun et al.,“Ceramidase Compositions and Methods Based Thereon”, PCT/US01/02866published as WO01/55410 on Aug. 2, 2001; Kimura et al., “Effect ofN,N,N,-trimethylsphingosine on Protein Kinase-C Activity; Melanoma CellGrowth In Vitro; Metastatic Potential In Vivo and Human PlateletAggregation”, Pat. No. 5,331,014, dated July 19, 1994; Igarashi et al.,“Effect of N,N,N,-trimethylsphingosine on Protein Kinase C ActivityMelanoma Cell Growth In Vitro; Metastatic Potential In Vivo and HumanPlatelet Aggregation”, U.S. Pat. No. 5,137,919 dated Aug. 11, 1992;Handa et al., “Effect of N,N,N,-trimethylsphingosine on Protein Kinase-CActivity, Melanoma Cell Growth In Vitro, Metastatic Potential In Vivoand Human Platelet Aggregation”, U.S. Pat. No., 5,151,360 dated Sep. 29,1992; Takesako et al., “Sphingosine Analogues”, PCT/JP98/01038,published as W098/40349 on Sep. 16, 1998; Kobori et al., “SphingosineDerivatives”, PCT/JP00/08229, published as WO01/38295 on May 31, 2001;Takesako et al., “Sphingosine Derivatives and Medicinal Composition”,PCT/JP98/04093, published as W099/12890 on Mar. 18, 1999; Liotta et al.,“Sphingolipid Derivatives and Their Methods of Use”, PCT/US99/03093,published as W099/41266 on Aug. 19, 1999; Macchia et al., “CeramideAnalogs, Process for their Preparation and their Use as AntitumorAgents”, PCT/EP00/07023, published as WO01/07418 on Feb. 1, 2001;Shayman et al., “Amino Ceramide-Like Compounds and Therapeutic Methodsof Use”, PCT/US00/1 8935, published as WO01/04108 on Jan. 18, 2001;Bielawska et al.,“(1S,2R)-D-erhthro-2-(N-Myristoylamino)-1-phenyl-1-propanol as anInhibitor of Ceramidase”, The Journal of Biological Chemistry, Vol. 271,May 24, 1996, pp. 12646-12654; Wanebo et al., “Ceramide andChemotherapeutic Agents for Inducing Cell Death”, PCT/US00/09440,published as WO00/59517 on Oct. 12, 2000; Ali et al., “CeramideDerivatives and Method of Use”, PCT/US01/09894, published as WO01/72701on Oct. 4, 2001; Eibl et al., “Pseudoceramides”, PCT/EP99/07698,published as WO00/21919 on Apr. 20, 2000; Jonghe et al.,“Structure-Activity Relationship of Short-Chain Sphingoid Bases AsInhibitors of Sphingosine Kinase”, Bioorganic & Medicinal ChemistryLetters 9:3175-3180, 1999; Arenz et al., “Synthese des ersten selektivenirreverilben Inhibitors der neutralen Sphingomyelinase”, Angew Chem.,112:1498-1500, 2000; and Abe et al., “Use of Sulfobutyl Ether-Cyclodextrin as a Vehicle forD-threo-1-Phenyl-2-decanoylamino-3-morpholinopropanol-RelatedGlucosylceramide Synthase Inhibitors”, Analytical Biochemistry,287:344-347, 2000.

[0213] One aspect of the invention involves identifying sphingolipidsthat are useful in sphingolipid-based therapy. This can done by testingcommercially available or otherwise obtainable sphingolipids in assaysthat measure the activity of enzymes involved in sphingolipid metabolismand/or intracellular signalling.

[0214] Commercially available sphingolipids (Avanti Polar Lipids, Inc.,Alabaster, AL) include without limitation synthetic D-erythro (C-18)derivatives of sphingosine, e.g., D-erythro Sphingosine (synthetic),Sphingosine-1-Phosphate, D-erythro Ceramide-1-Phosphate,N,N-Dimethylsphingosine, N,N,N-Trimethylsphingosine,Sphingosylphosphorylcholine, Sphingomyelin, and Ceramides; D-erythro(C-18) derivatives of sphinganine (dihydrosphingosine), e.g.,Sphinganine-1-Phosphate, D-erythro Sphinganine, N-Acyl-Sphinganine C2,N-Acyl-Sphinganine C8, N-acyl-Sphinganine C16, N-Acyl-Sphinganine C18,N-Acyl-Sphinganine C24, and N-Acyl-Sphinganine C24:1; glycosylated (C18) sphingosine and phospholipid derivatives, e.g., glycosylatedsphingosine, ceramide and phosphatidylethanolamine, betaD-glucosyl-sphingosine, and beta D-galactosyl-sphingosine; D-erythro(C17) derivatives, e.g., D-erythro Sphingosine and D-erythroSphingosine-1-phosphate; D-erythro (C20) derivatives, such as D-erythrosphingosine; and L-threro (C18) derivatives such as L-threo Sphingosineand L-threo Dihydrosphingosine (Safingol). Phytosphingosine derivativesfrom yeast. e.g., Phytosphingosine, D-ribo-Phytosphingosine-1-Phosphate,N-Acyl Phytosphingosine C2, N-Acyl Phytosphingosine C8 and N-AcylPhytosphingosine C18 may also be used.

[0215] A variety of methods for synthesizing sphingolipids andsphingolipid-related molecules are known. In addition to the above-citedreferences, see Szulc et al., “A facile regioselective synthesis ofsphingosine 1-phosphate and ceramide 1-phosphate, Tetrahedron Letter41:7821-7824, 2000; Igarashi et al., ” Sphingosine-1-PhosphateEssentially Free of L-Threo Isomer, U.S. Pat. No. 5,663,404, issued Sep.2, 1997; Boumendjel et al., “Method For Preparation of Sphingoid Bases”,U.S. Pat. No. 5,430,160, issued Jul. 4, 1995; Ito et al., “Process forthe Preparation of Sphingolipids and Sphingolipid Derivatives”,PCT/JP97/02483, published as WO98/03529 on Jan. 29, 1998; and Igarashiet al., “Method of Preparing N,N,N,-trimethylsphingosine”, Pat. No.5,248,824 dated Sep. 28, 1993.

[0216] In a preferred embodiment, sphingolipids having a desiredactivity are identified by high throughput screening (HTS) ofcombinatorial libraries of sphingolipid-related compounds. Combinatorialsphingolipid libraries are prepared according to methods known in theart, or may be purchased commercially. One type of combinatorialsphingolipid library that may be used is the BIOMOL Bioactive LipidLibrary (Affiniti Research Products Ltd., Mamhead, U.K.).

[0217] High Throughput Screening (HTS) Assays

[0218] HTS typically uses automated assays to search through largenumbers of compounds for a desired activity. Typically HTS assays areused to find new drugs by screening for chemicals that act on aparticular enzyme or molecule. For example, if a chemical inactivates anenzyme it might prove to be effective in preventing a process in a cellwhich causes a disease. High throughput methods enable researchers totry out thousands of different chemicals against each target veryquickly using robotic handling systems and automated analysis ofresults.

[0219] As used herein, “high throughput screening” or “HTS” refers tothe rapid in vitro screening of large numbers of compounds (libraries);generally tens to hundreds of thousands of compounds, using roboticscreening assays. Ultra high-throughput Screening (UHTS) generallyrefers to the high-throughput screening accelerated to greater than100,000 tests per day.

[0220] To achieve high-throughput screening, it is best to house sampleson a multicontainer carrier or platform. A multicontainer carrierfacilitates measuring reactions of a plurality of candidate compoundssimultaneously. Multi-well microplates may be used as the carrier. Suchmulti-well microplates, and methods for their use in numerous assays,are both known in the art and commercially available.

[0221] Screening assays may include controls for purposes of calibrationand confirmation of proper manipulation of the components of the assay.Blank wells that contain all of the reactants but no member of thechemical library are usually included. As another example, a knowninhibitor (or activator) of an enzyme for which modulators are sought,can be incubated with one sample of the assay, and the resultingdecrease (or increase) in the enzyme activity determined according tothe methods herein. It will be appreciated that modulators can also becombined with the enzyme activators or inhibitors to find modulatorswhich inhibit the enzyme activation or repression that is otherwisecaused by the presence of the known the enzyme modulator. Similarly,when ligands to a sphingolipid target are sought, known ligands of thetarget can be present in control/calibration assay wells.

[0222] Measuring Enzymatic and Binding Reactions During Screening Assays

[0223] Techniques for measuring the progression of enzymatic and bindingreactions in multicontainer carriers are known in the art and include,but are not limited to, the following.

[0224] Spectrophotometric and spectrofluorometric assays are well knownin the art. Examples of such assays include the use of calorimetricassays for the detection of peroxides, as disclosed in Example 1(b) andGordon, A. J. and Ford, R. A., The Chemist's Companion: A Handbook OfPractical Data, Techniques, And References, John Wiley and Sons, N.Y.,1972, Page 437.

[0225] Fluorescence spectrometry may be used to monitor the generationof reaction products. Fluorescence methodology is generally moresensitive than the absorption methodology. The use of fluorescent probesis well known to those skilled in the art. For reviews, see Bashford etal., Spectrophotometry and Spectrofluorometry: A Practical Approach, pp.91-114, IRL Press Ltd. (1987); and Bell, Spectroscopy In Biochemistry,Vol. 1, pp. 155-194, CRC Press (1981).

[0226] In spectrofluorometric methods, enzymes are exposed to substratesthat change their intrinsic fluorescence when processed by the targetenzyme. Typically, the substrate is nonfluorescent and converted to afluorophore through one or more reactions. As a non-limiting example,SMase activity can be detected using the Amplex® Red reagent (MolecularProbes, Eugene, OR). In order to measure sphingomyelinase activity usingAmplex Red, the following reactions occur. First, SMase hydrolyzessphingomyelin to yield ceramide and phosphorylcholine. Second, alkalinephosphatase hydrolyzes phosphorylcholine to yield choline. Third,choline is oxidized by choline oxidase to betaine. Finally, H202, in thepresence of horseradish peroxidase, reacts with Amplex Red to producethe fluorescent product, Resorufin, and the signal therefrom is detectedusing spectrofluorometry.

[0227] Fluorescence polarization (FP) is based on a decrease in thespeed of molecular rotation of a fluorophore that occurs upon binding toa larger molecule, such as a receptor protein, allowing for polarizedfluorescent emission by the bound ligand. FP is empirically determinedby measuring the vertical and horizontal components of fluorophoreemission following excitation with plane polarized light. Polarizedemission is increased when the molecular rotation of a fluorophore isreduced. A fluorophore produces a larger polarized signal when it isbound to a larger molecule (i.e. a receptor), slowing molecular rotationof the fluorophore. The magnitude of the polarized signal relatesquantitatively to the extent of fluorescent ligand binding. Accordingly,polarization of the “bound” signal depends on maintenance of highaffinity binding.

[0228] FP is a homogeneous technology and reactions are very rapid,taking seconds to minutes to reach equilibrium. The reagents are stable,and large batches may be prepared, resulting in high reproducibility.Because of these properties, FP has proven to be highly automatable,often performed with a single incubation with a single, premixed,tracer-receptor reagent. For a eview, see Owickiet al., Application ofFluorescence Polarization Assays in High-Throughput Screening, GeneticEngineering News, 17:27, 1997.

[0229] FP is particularly desirable since its readout is independent ofthe emission intensity (Checovich, W. J., et al., Nature 375:254-256,1995; Dandliker, W. B., et al., Methods in Enzymology 74:3-28, 1981) andis thus insensitive to the presence of colored compounds that quenchfluorescence emission. FP and FRET (see below) are well-suited foridentifying compounds that block interactions between sphingolipidreceptors and their ligands. See, for example, Parker et al.,Development of high throughput screening assays using fluorescencepolarization: nuclear receptor-ligand-binding and kinase/phosphataseassays, J. Biomol Screen 5:77-88, 2000.

[0230] Fluorophores derived from sphingolipids that may be used in FPassays are commercially available. For example, Molecular Probes(Eugune, OR) currently sells sphingomyelin and one ceramide flurophores.These are, respectively,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosylphosphocholine (BODIPY® FL C5-sphingomyelin);N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosylphosphocholine (BODIPY® FL C12-sphingomyelin); andN-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine(BODIPY® FL C5-ceramide). U.S. Pat. No. 4,150,949, (Immunoassay forgentamicin), discloses fluorescein-labelled gentamicins, includingfluoresceinthiocarbanyl gentamicin. Additional fluorophores may beprepared using methods well known to the skilled artisan.

[0231] Exemplary normal-and-polarized fluorescence readers include thePOLARION fluorescence polarization system (Tecan AG, Hombrechtikon,Switzerland). General multiwell plate readers for other assays areavailable, such as the VERSAMAX reader and the SPECTRAMAX multiwellplate spectrophotometer (both from Molecular Devices).

[0232] Fluorescence resonance energy transfer (FRET) is another usefulassay for detecting interaction and has been described previously. See,e.g., Heim et al., Curr. Biol. 6:178-182, 1996; Mitra et al., Gene173:13-17 1996; and Selvin et al., Meth. Enzymol. 246:300-345, 1995.FRET detects the transfer of energy between two fluorescent substancesin close proximity, having known excitation and emission wavelengths. Asan example, a protein can be expressed as a fusion protein with greenfluorescent protein (GFP). When two fluorescent proteins are inproximity, such as when a protein specifically interacts with a targetmolecule, the resonance energy can be transferred from one excitedmolecule to the other. As a result, the emission spectrum of the sampleshifts, which can be measured by a fluorometer, such as a fMAX multiwellfluorometer (Molecular Devices, Sunnyvale Calif.).

[0233] Scintillation proximity assay (SPA) is a particularly usefulassay for detecting an interaction with the target molecule. SPA iswidely used in the pharmaceutical industry and has been described(Hanselman et al., J. Lipid Res. 38:2365-2373 (1997); Kahl et al., Anal.Biochem. 243:282-283 (1996); Undenfriend et al., Anal. Biochem.161:494-500 (1987)). See also U.S. Pat. Nos. 4,626,513 and 4,568,649,and European Pat. No. 0,154,734. One commercially available system usesFLASHPLATE scintillant-coated plates (NEN Life Science Products, Boston,Mass.).

[0234] The target molecule can be bound to the scintillator plates by avariety of well known means. Scintillant plates are available that arederivatized to bind to fusion proteins such as GST, His6 or Flag fusionproteins. Where the target molecule is a protein complex or a multimer,one protein or subunit can be attached to the plate first, then theother components of the complex added later under binding conditions,resulting in a bound complex.

[0235] In a typical SPA assay, the gene products in the expression poolwill have been radiolabeled and added to the wells, and allowed tointeract with the solid phase, which is the immobilized target moleculeand scintillant coating in the wells. The assay can be measuredimmediately or allowed to reach equilibrium. Either way, when aradiolabel becomes sufficiently close to the scintillant coating, itproduces a signal detectable by a device such as a TOPCOUNT NXTmicroplate scintillation counter (Packard BioScience Co., MeridenConn.). If a radiolabeled expression product binds to the targetmolecule, the radiolabel remains in proximity to the scintillant longenough to produce a detectable signal.

[0236] In contrast, the labeled proteins that do not bind to the targetmolecule, or bind only briefly, will not remain near the scintillantlong enough to produce a signal above background. Any time spent nearthe scintillant caused by random Brownian motion will also not result ina significant amount of signal. Likewise, residual unincorporatedradiolabel used during the expression step may be present, but will notgenerate significant signal because it will be in solution rather thaninteracting with the target molecule. These non-binding interactionswill therefore cause a certain level of background signal that can bemathematically removed. If too many signals are obtained, salt or othermodifiers can be added directly to the assay plates until the desiredspecificity is obtained (Nichols et al., Anal. Biochem. 257:112-119,1998).

[0237] Assays for Enzymes Involved in Sphingolipid Metabolism

[0238] SMase: A variety of methods are available to measure SMaseactivity. It is possible to assay the SMase activity in vivo throughlabeling the cells with a radioactive substrate for SMase and thendetermining the level of radiolabel in the enzymatic products. Liu, B.,and Y. A. Hannun. “Sphingomyelinase Assay Using Radiolabeled Substrate.”Sphingolipid Metabolism and Cell Signaling, Pt a., 164-67. Methods inEnzymology, vol. 311, 2000. HTS assays of SMase activity are describedby Lin et al., “Sphingomyelinase Assay Using Radiolabeled Substrate”, p.164, Liu et al., Barbone et al., “Robotic Assay of Sphingomyelinase forHigh-Throughput Screening”, p. 168, and Hessler et al., “A HighThroughput Sphingomyelinase Assay”, p. 176 In: Sphingolipid Metabolismand Cell Signaling, Hannun, Yusaf A. (editor); Merrill, Alfred H.(editor), Academic Press (1999). The activity of SMase can be alsodetermined in vitro either using radiolabelled SM, or a chromogenicanalog of SM or colored and fluorescent derivatives of natural SM.Torley et al. (A turbidometric assay for phospholipase C andsphingomyelinase, Anal Biochem 222:461-464, 1994) describe aturbidometric assay for SMase suitable for high-volume screening usingunmodified substrates.

[0239] SPH Kinase: Assays for SPH kinase are described by Olivera etal., “Assaying Sphingosine Kinase Activity”, Methods in Enzymology,311:215-223, 1999; and Caligan et al., “A High-Performance LiquidChromatographic Method to Measure Sphingosine 1-Phosphate and RelatedCompounds from Sphingosine Kinase Assays and Other Biological Samples”,Analytical Biochemistry, 281:36-44, 2000.

[0240] CER Kinase: Assays for CER kinase are disclosed by Bajjalieh etal., “Ceramide Kinase”, Methods in Enzymology, 311:207-215, 1999.

[0241] Ceramidase: Assays for Ceramidase are disclosed by Zhang et al.,“Human Acid Ceramidase Gene: Novel Mutations in Farber Disease”,Molecular Geneetics and Metabolism, 70:301-309, 2000.

[0242] CER Synthase: Assays for CER synthase are disclosed byBose etal., “Measurement of Ceramide Synthase Activity”, Methods in Enzymology,322:378-382,2000.

[0243] Glucosylceramide Synthase: Assays for glucosylceramide synthaseare disclosed by Shayman et al., “Glucosylceramide Synthase: Assay andProperties”, Methods in Enzymology, 311:42-49, 1999.

[0244] Assays for Sphingolipids:

[0245] S-1-P assays are disclosed by Ruwisch et al., “An improvedhigh-performance liquid chromatographic method for the determination ofsphingosine-1-phosphate in complex biological materials”,Naunyn-Schmiedeberg's Arch Pharmacol, 363:358-363, 2001; Edsall et al.,“Enzymatic Measurement of Sphingosine 1-Phosphate”, AnalyticalBiochemistry, 272:80-86, 1999; and Edsall et al., “Enzymatic Method forMeasurement of Sphingosine 1-Phosphate”, Methods in Enzymology,312:9-16, 2000.

[0246] SPH assays are disclosed by Chmura et al. (Down-Regulation ofCeramide Production Abrogates Ionizing Radiation-Induced Cytochrome cRelease and Apoptosis, Molecular Pharmacology, 57:792-796, 2001); U.S.Pat. No. 5,677,189, and Shephard et al. (Liquid chormatographicdetermination of the sphinganine/sphingosine ratio in serum, Journal ofChromatography B, 710:291-222, 1998).

[0247] Pharmaceutical Compositions

[0248] Another aspect of the invention is drawn to compositions,including but not limited to pharmaceutical and/or biologicalcompositions. According to the invention, a “composition” refers to amixture comprising at least one carrier, preferably a physiologicallyacceptable carrier, and one or more therapeutic agents according to theinvention. The term “carrier” defines a chemical compound that does notinhibit or prevent the incorporation of therapeutic agents into cells ortissues. A carrier typically is an inert substance that allows an activeingredient to be formulated or compounded into a suitable dosage form(e.g., a pill, a capsule, a gel, a film, a tablet, a microparticle(e.g., a microsphere), a solution etc.). A “physiologically acceptablecarrier” is a carrier suitable for use under physiological conditionsthat does not abrogate (reduce, inhibit, or prevent) the biologicalactivity and properties of the compound. For example, dimethyl sulfoxide(DMSO) is a carrier as it facilitates the uptake of many organiccompounds into the cells or tissues of an organism. Preferably, thecarrier is a physiologically acceptable carrier, preferably apharmaceutically or veterinarily acceptable carrier, in which thetherapeutic agent is disposed. A “pharmaceutical composition” refers toa composition wherein the carrier is a pharmaceutically acceptablecarrier, while a “veterinary composition” is one wherein the carrier isa veterinarily acceptable carrier. The term “pharmaceutically acceptablecarrier” or “veterinarily acceptable carrier” includes any medium ormaterial that is not biologically or otherwise undesirable, i.e., thecarrier may be administered to an organism along with a therapeuticagent, composition or compound without causing any undesirablebiological effects or interacting in a deleterious manner with thecomplex or any of its components or the organism. Examples ofpharmaceutically acceptable reagents are provided in The United StatesPharmacopeia, The National Formulary, United States PharmacopeialConvention, Inc., Rockville, Md. 1990, hereby incorporated by referenceherein into the present application. The terms “therapeuticallyeffective amount” or “pharmaceutically effective amount” mean an amountsufficient to induce or effectuate a measurable response in the targetcell, tissue, or organism. What constitutes a therapeutically effectiveamount will depend on a variety of factors which the knowledgeablepractitioner will take into account in arriving at the desired dosageregimen.

[0249] The compositions of the invention can further comprise otherchemical components, such as diluents and excipients. A “diluent” is achemical compound diluted in a solvent, preferably an aqueous solvent,that facilitates dissolution of the therapeutic agent in the solvent,and it may also serve to stabilize the biologically active form of thetherapeutic agent or one or more of its components. Salts dissolved inbuffered solutions are utilized as diluents in the art. For example,preferred diluents are buffered solutions containing one or moredifferent salts. A preferred buffered solution is phosphate bufferedsaline (particularly in conjunction with compositions intended forpharmaceutical administration), as it mimics the salt conditions ofhuman blood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a therapeutic agent.

[0250] An “excipient” is any more or less inert substance that can beadded to a composition in order to confer a suitable property, forexample, a suitable consistency or to form a drug. Suitable excipientsand carriers include, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol cellulose preparations such as,for example, maize starch, wheat starch, rice starch, agar, pectin,xanthan gum, guar gum, locust bean gum, hyaluronic acid, casein potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, polyacrylate, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents can also be included, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. Other suitable excipients and carriers includehydrogels, gellable hydrocolloids, and chitosan. Chitosan microspheresand microcapsules can be used as carriers. See WO 98/52547 (whichdescribes microsphere formulations for targeting compounds to thestomach, the formulations comprising an inner core (optionally includinga gelled hydrocolloid) containing one or more active ingredients, amembrane comprised of a water insoluble polymer (e.g., ethylcellulose)to control the release rate of the active ingredient(s), and an outerlayer comprised of a bioadhesive cationic polymer, for example, acationic polysaccharide, a cationic protein, and/or a synthetic cationicpolymer; U.S. Pat. No. 4,895,724. Typically, chitosan is cross-linkedusing a suitable agent, for example, glutaraldehyde, glyoxal,epichlorohydrin, and succinaldehyde. Compositions employing chitosan asa carrier can be formulated into a variety of dosage forms, includingpills, tablets, microparticles, and microspheres, including thoseproviding for controlled release of the active ingredient(s). Othersuitable bioadhesive cationic polymers include acidic gelatin,polygalactosamine, polyamino acids such as polylysine, polyhistidine,polyornithine, polyquaternary compounds, prolamine, polyimine,diethylaminoethyldextran (DEAE), DEAE-imine, DEAE-methacrylate,DEAE-acrylamide, DEAE-dextran, DEAE-cellulose, poly-p-aminostyrene,polyoxethane, copolymethacrylates, polyamidoamines, cationic starches,polyvinylpyridine, and polythiodiethylaminomethylethylene.

[0251] The compositions of the invention can be formulated in anysuitable manner. Therapeutic agents may be uniformly (homogeneously) ornon-uniformly (heterogeneously) dispersed in the carrier. Suitableformulations include dry and liquid formulations. Dry formulationsinclude freeze dried and lyophilized powders, which are particularlywell suited for aerosol delivery to the sinuses or lung, or for longterm storage followed by reconstitution in a suitable diluent prior toadministration. Other preferred dry formulations include those wherein acomposition according to the invention is compressed into tablet or pillform suitable for oral administration or compounded into a sustainedrelease formulation. When the composition is intended for oraladministration but the therapeutic agent is to be delivered toepithelium in the intestines, it is preferred that the formulation beencapsulated with an enteric coating to protect the formulation andprevent premature release of the therapeutic agents included therein. Asthose in the art will appreciate, the compositions of the invention canbe placed into any suitable dosage form. Pills and tablets representsome of such dosage forms. The compositions can also be encapsulatedinto any suitable capsule or other coating material, for example, bycompression, dipping, pan coating, spray drying, etc. Suitable capsulesinclude those made from gelatin and starch. In turn, such capsules canbe coated with one or more additional materials, for example, andenteric coating, if desired. Liquid formulations include aqueousformulations, gels, and emulsions.

[0252] In one embodiment, the pharmaceutical composition is formulatedfor rapid cardiac delivery. One type of pharmaceutical composition thatis formulated for rapid cardiac delivery is an injectable pharmaceuticalcomposition. Liquid pharmaceutical compositions which are sterilesolutions or suspensions can be utilized by for example, intramuscular,intrathecal, epidural, intraperitoneal or subcutaneous injection.Sterile solutions can also be administered intravenously. The activeingredient may be prepared as a sterile solid composition which may bedissolved or suspended at the time of administration using sterilewater, saline, or other appropriate sterile injectable medium. Carriersare intended to include necessary and inert binders, suspending agents,lubricants, flavorants, sweeteners, preservatives, dyes, and coatings.

[0253] Some preferred embodiments concern compositions that comprise abioadhesive, preferably a mucoadhesive, coating. A “bioadhesive coating”is a coating that allows a substance (e.g., a composition or therapeuticagent according to the invention) to adhere to a biological surface orsubstance better than occurs absent the coating. A “mucoadhesivecoating” is a preferred bioadhesive coating that allows a substance, forexample, a composition according to the invention, to adhere better tomucosa occurs absent the coating. For example, micronized particles(e.g., particles having a mean diameter of about 0.01, 0.1, 0.2, 0.3,0.4, 0.5, 1, 5, 10, 25, 50, or 100 um) can be coated with amucoadhesive. In instances wherein the therapeutic agent is a solublemolecule, including but not limited to soluble receptor fragments,antibodies and antibody derivatives or other soluble polypeptides,preferred diameters include but are not limited to about 0.1, 0.3, 0.5,0.6, 0.7, 0.8, 0.9, and 1.0 um. In instances where the therapeutic agentis a soluble molecule, including but nor limited to soluble receptorfragments and derivatives, antibodies and antibody derivatives and othersoluble polypeptides, preferred diameters are about 0.1, 0.3, 0.5, 0.6,0.7, 0.8, 0.9 and 1.0 um. The coated particles can then be assembledinto a dosage form suitable for delivery to an organism. Preferably, anddepending upon the location where the cell surface transport moiety tobe targeted is -expressed, the dosage form is then coated with anothercoating to protect the formulation until it reaches the desiredlocation, where the mucoadhesive enables the formulation to be retainedwhile the therapeutic agents interact with the target cell surfacetransport moiety.

[0254] Those skilled in the art will appreciate that when thecompositions of the present invention are administered as agents toachieve a particular desired biological result, which may include atherapeutic or protective effect(s) (including vaccination), it may benecessary to combine the therapeutic agents of the invention with asuitable pharmaceutical carrier. The choice of pharmaceutical carrierand the preparation of the therapeutic agent as a therapeutic orprotective agent will depend on the intended use and mode ofadministration. Suitable formulations and methods of administration oftherapeutic agents include those for oral, pulmonary, nasal, buccal,occular, dermal, rectal, or vaginal delivery.

[0255] Depending on the mode of delivery employed, the context-dependentfunctional entity can be delivered in a variety of pharmaceuticallyacceptable forms. For example, the context-dependent functional entitycan be delivered in the form of a solid, solution, emulsion, dispersion,micelle, liposome, and the like, incorporated into a pill, capsule,tablet, suppository, aerosol, droplet, or spray. Pills, tablets,suppositories, aerosols, powders, droplets, and sprays may have complex,multilayer structures and have a large range of sizes. Aerosols,powders, droplets, and sprays may range from small (1 micron) to large(200 micron) in size.

[0256] Pharmaceutical compositions of the present invention can be usedin the form of a solid, a solution, an emulsion, a dispersion, amicelle, a liposome, and the like, wherein the resulting compositioncontains one or more of the compounds of the present invention, as anactive ingredient, in admixture with an organic or inorganic carrier orexcipient suitable for enteral or parenteral applications. The activeingredient may be compounded, for example, with the usual non-toxic,pharmaceutically acceptable carriers for tablets, pellets, capsules,suppositories, solutions, emulsions, suspensions, and any other formsuitable for use. The carriers which can be used include glucose,lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, medium chain length triglycerides, dextrans, and othercarriers suitable for use in manufacturing preparations, in solid,semisolid, or liquid form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used. Examples of astabilizing dry agent includes triulose, preferably at concentrations of0.1% or greater (See, e.g., U.S. Pat. No. 5,314,695).

[0257] Pharmaceutical compositions that preferentially target cardiactissues are generally preferred. As a non-limiting example, U.S. Pat.No. 5,876,747 to Stracher et al. claims liposomes that preferentiallytravel to cardiac and skeletal muscle. Medical Devices and Kits Medicaldevices that incorporate the therapeutic agents of the invention may beprepared and used. Such devices and kits may be designed for use bytrained medical personnel in, e.g., hospitals, ambulances, and the like.Additionally or alternatively, such devices and kits may be designed tobe used by untrained individuals, including a patient in need oftreatment, in situations where trained medical personnel are notavailable. Non-limiting examples of such devices and kits are disclosedin U.S. Pat. No. 4,658,830 to Sarnoff. Such devices and kits may furtherinclude other (supplementary) devices and formulations useful fortreating cardiac disorders. A non-limiting example of one suchsupplementary device is a portable defibrillator and similar devices,such as is disclosed in U.S. Pat. RE 30,750 to Diack et al. Anon-limiting example of a supplementary formulation is one that includescompounds useful for treating conditions associated with cardiacdisorders, such as those that are disclosed in U.S. Pat. No. 6,130,235to Mavunkel et al.

[0258] Medical devices that incorporate sphingolipid-binding ligands,such as antibodies, according to the invention include those that arecommonly referred to as “dialysis machines” (see, e.g., U.S. Pat. No.6,080,321). In this type of sphingolipid-based cardiac medical device,sphingolipid ligands are immobilized onto a surface in the device. Apatient's blood is pumped into the device in such a way as to bringblood into contact with the sphingolipid ligands. Undesirable, toxicand/or cardiotoxic sphingolipids and/or their metabolic precursors inthe blood bind to the immobilized sphingolipid ligands, thus removingundesirable, toxic and/or cardiotoxic sphingolipids and/or their lesstoxic metabolic precursors from the blood. The blood, which is returnedto the patient after passing through the device, leaves the device whilethe targeted sphingolipid remains bound to, and thus retained by, theimmobilized sphingolipid ligands. The patient's blood re-enters thepatient's body relatively or completely depleted of the targetedsphingolipid. The passage of a patient's blood through the device isrepeated as many times as is needed in order to achieve the desiredeffect of lowering the concentration of undesirable, toxic and/orcardiotoxic sphingolipids and their metabolic precursors.

[0259] An in-dwelling catheter would be inserted to the area at risk inthe heart. The catheter would have a smaller inner catheter which wouldsubsequently inserted. The smaller catheter would be coated with anantibody or other sphingolipid binding ligands which would act as a sinkto remove the sphingolipids in the focused area. This leads to alocalized depletion or complete removal of undesirable sphingolipids.

[0260] The invention provides for diagnostic and therapeutic kitsrelated to sphingolipid-based therapy. In one embodiment, the inventionrelates to kits for determining the diagnosis or prognosis of a patient.These kits preferably comprise devices and reagents for measuring one ormore marker levels in a test sample from a patient, and instructions forperforming the assay. Optionally, the kits may contain one or more meansfor converting marker level(s) to a prognosis. Such kits preferablycontain sufficient reagents to perform one or more such determinations.

[0261] More specifically, a diagnostic kit of the invention comprisesany of the following reagents and/or components in any combination.

[0262] 1. A detectable or detectably labeled first reagent that binds aspingolipid or spingolipid metabolite of interest. Thesphingolipid-binding reagent can, but need not, be an antibody or anantibody derivative comprising a detectable moiety. Thesphingolipid-binding reagent is stored in an openable container in thekit, or is bound to a surface of a substrate such that it is accessibleto other reagents. Examples of the latter include test strips.

[0263] 2. If the first reagent in neither detectable nor detectablylabeled, the kit may comprise a detectable or detectably labeled secondreagent that binds to the first reagent (e.g., a secondary antibody) orwhich produces a detectable signal when in close proximity to the firstreagent (e.g., as results from fluorescent resonance energy transferFRET). In either case, the signal produced from the second reagentcorrelates with the amount of sphingolipid in the sample.

[0264] 3. One or more positive control reagents. Typically, thesereagents comprise a compound that is known to produce a signal in theassay. In one embodiment, the positive control reagents are standards,i.e., comprise a known amount of a detectable or detectably labeledcompound, the signal from which may be compared to the signal from atest sample. In addition to serving as positive control reagents, theymay be used to develop calibration curves that relate the amount ofsignal to the known concentration of a detectable or detectably labeledcompound. The signal from a test sample is compared to the calibrationcurve in order to determine what concentration of the detectable ordetectably labeled compound corresponds to the signal from the testsample. In this embodiment, the kit provides quantitative measurementsof the amount of a sphingolipid in a test sample.

[0265] 4. One or more negative control reagents. Typically, thesecontrol reagents may comprise buffer or another solution that does notcontain any of the detectable or detectably labeled first or secondreagents and should thus not produce any detectable signal. Any signalthat is detected reflects the background level of “noise” in the assay.Another type of negative control reagent contains most of the componentsnecessary for the signal of the assay to be produced, but lacks at leastone such component and therefor should not produce a signal. Yet anothertype of negative control reagent contains all of the componentsnecessary for the signal of the assay to be produced, but also containsan inhibitor of the process that produced the signal.

[0266] 5. One or more auxiliary reagents for use in the diagnosticassays of the kit, e.g., buffers, alcohols, acid solutions, etc. Thesereagents are generally available in medical facilities and thus areoptional components of the kit. However, these reagents preferably areincluded in the kit to ensure that reagents of sufficient purity andsterility are used, since the resulting protein conjugates are to beadministered to mammals, including humans, for medical purposes, and toprovide kits that can be used in situations where medical facilities arenot readily available, e.g., when hiking in places located far frommedical facilities, or in situations where the presence of theseauxiliary reagents allows for the immediate treatment of a patientoutside of a medical facility as opposed to treatment that arrives atsome later time (e.g.,

[0267] 6. Instructions to a person using a kit for its use. Theinstructions can be present on one or more of the kit components, thekit packaging and/or a kit package insert.

[0268] A therapeutic kit of the invention comprises any of the followingreagents and/or components in any combination.

[0269] 1. One or more therapeutic agents.

[0270] 2. If the therapeutic agent(s) are not formulated for deliveryvia the alimentary canal, which includes but is not limited tosublingual delivery, a device capable of delivering the therapeuticagent through some other routes. One type of device for parenteraldelivery is a syringe that is used to inject the therapeutic agent intothe body of an animal in need of the therapeutic agent. Inhalationdevices may also be used. A device for delivering gentamicin to apatient via inhalation is disclosed in U.S. patent

[0271] 3. Separate containers, each of which comprises one or morereagents of the kit. In a preferred embodiment, the containers are vialscontain sterile, lyophilized formulations of a therapeutic compositionthat are suitable for reconstitution.

[0272] 4. Instructions to a person using a kit for its use. Theinstructions can be present on one or more of the kit components, thekit packaging and/or a kit package insert.

[0273] For a better understanding of the present invention, reference ismade to the accompanying drawings and detailed description and its scopewill be pointed out in the appended claims. All references cited hereinare hereby incorporated by reference.

EXAMPLES

[0274] The following examples are non-limiting and are merelyrepresentative of various aspects and features of the present invention.

Example 1

[0275] Sphingosine Production in Rabbits Increases in Cardiac Ischemia

[0276] Tissue levels of sphingosine (SPH) in adult rabbit hearts undervarious conditions were determined as follows. Rabbits were subjected toretrograde coronary perfusion with hypoxic (low oxygen) conditions(i.e., 95% CO₂; 5% O₂) or normal Kreb's buffers (equilibrated with 95%O₂; 5% CO₂). The rabbits were sacrificed, and hearts were removed andquickly homogenized. Sphingolipids were extracted from homogenates usingprotocols essentially as described by Sabbadini et al., Biochem.Biophys. Res. Comm. 193:752-758, 1993. HPLC analysis of the extractedlipids revealed significant increases in tissue SPH levels for heartsperfused with CO₂ when compared to hearts exposed to control conditions(20-fold, p<0.001). These increases occurred after only 5 minutes ofhypoxia.

Example 2

[0277] Sphingosine Production in Humans

[0278] Serum levels of SPH levels in human patients experiencing cardiacischemia were examined as follows. Serum samples were taken frompatients presenting themselves to the emergency department of the NavalMedical Center of San Diego using strict human subjects protocols.Sphingolipids in the blood samples were extracted and analyzed asdescribed above. Patients with confirmed myocardial ischemia hadsignificantly higher SPH levels than any of the control groups.

[0279] Serum SPH levels in six “well-conditioned athletes” were combinedas one control group. These subjects were Navy special forces (NavySeals) and Olympic athletes from the Olympic Training Center who wereexercised to exhaustion on treadmills at 49° C. In the case of theOlympic athletes, individuals at rest were also evaluated. Thewell-conditioned athletic control group had serum SPH levels of 4.18±1.8pmol/mL, ranging from the lower limit of detection (5 pmol/mL) to 16.4pmol/mL.

[0280] An age-matched (47-79 yrs) control group consisted of fifteensubjects enrolled in the San Diego State University (SDSU) Adult FitnessProgram who tested negative for exercise stress (treadmill) and nosymptoms of acute coronary syndrome (e.g., chest pain). The age-matchedcontrol group had a mean serum level of SPH of 99.3±32.4 pmol/mL,ranging from the lower limit of detection to 369 pmol/mL.

[0281] A group of ischemic patients (n=9) were individuals who hadmyocardial ischemia, tested positive for exercise stress for exercisetreadmill testing and/or were referred to the catherization lab forpercutaneous coronary revascularization (angioplasty). The ischemicpatients had a mean serum SPH level of 697±80.7 pmol/mL.

[0282] The ischemic patient subgroup with angina symptoms who underwentangioplasty had average pre-procedure serum SPH levels of 885±123pmol/mL (ranging from 447 to 1122 pmol/mL). The patient serum SPH levelswere significantly (p<0.001) higher than the SPH levels of theage-matched control group. When the control group of ischemic patientswas examined, an average serum SPH level of 697±80.7 pmol/mL wasobtained. This value is ˜7-fold higher than the age-matched controlgroup (p<0.001) and ˜160-fold higher than well-conditioned athletes.

Example 3

[0283] Hypoxic Effects on the Sphingomylein Signal Transduction Cascade

[0284] TNFα, acting via TNFα receptors, has been shown to utilize thesphingomyelin signal transduction cascade in cardiomyocytes (Oral etal., J. Biol. Chem. 272:4836-4842, 1997; Krown et al., J. Clin. Invest.98:2854-2865, 1996). The following experiments were carried out in orderto determine if this signaling system could also be activated byhypoxia, and if sphingolipids were produced before cell death as is thecase for TNFα, as described in the Detailed Description of theInvention.

[0285] Adult rat cardiomyocytes were subjected to hypoxic conditions asdescribed above and assayed for their ability to produce sphingolipidbases involved in the sphingomyelin signal transduction cascade.

[0286] Cardiomyocytes were cultured on plastic dishes that were placedin a humidified modular incubator chamber (ICN Biomedicals, Aurora,Ohio.) pressurized to 0.04-0.05 psi by the particular gas mixture usedin the treatment. Warm (37° C.) Tyrode's solution containing 0.2 mM BSA,ampicillin (50 mg/mL), kanamycin (100 mg/mL) and fungizone (20 μg/mL)was gassed in a 50 mL sterile conical for 15 minutes with 95%N₂/5% CO₂prior to adding the solution to the cultured cells. The pO₂ wasmonitored by a Micro pO₂ System oxygen electrode (Lasar Research Labs,Los Angeles, Calif.) and found to be 4.0 PPM for the duration of thehypoxia condition. The chamber was maintained at 37° C. for thedesignated times. Control (normoxia) cells were treated the same exceptfor the use of 95%O₂/5% CO₂ and incubated in a standard incubator. ThepO₂ of the normoxia treatment was 7.3 PPM. The pH of control andexperimental cell cultures was monitored with a micro pH electrode(Beetrode pH Electrodes, Sarasota, Fla.) and remained constant atpH=7.26 +/−0.02 for normoxic cells and 7.15+/−0.03 for hypoxic cellsthroughout the experiment. Because the cells were incubated in Tyrode'slacking glucose, these experiments represent a model of hypoxia withmetabolic inhibition. In selected experiments with neonatal ratcardiomyocytes, a third treatment following hypoxia was employed tosimulate reperfusion. This treatment consisted of 5 hours ofreoxygenation with 95%O₂/5% CO₂ in the incubation chamber. At the end ofeach incubation period, the cell-conditioned media was aspirated andsaved for analysis. In selected experiments, the cells were scraped inthe presence of 800 μl 1-butanol for the determination of both cellularand extracellular (cell-conditioned media) content of sphingolipids.

[0287] For the extraction of sphingolipids, 100-300 μl samples oftissue, serum or cell extracts were deproteinized by adding warmedbutanol (70° C., 800 μl), vortexing and incubating at 70° C. whilerocking. The mixture was then placed in a sonicating water bath for 10minutes. Denatured protein and aqueous phase were separated from thebutanol layer by centrifugation at 15,300×g. The upper butanol layer wastransferred into a new extraction tube and saponified by the addition0.5 M KOH (200 μl). After vortexing, samples were incubated at 70° C.while rocking for 1 hour with intermittent vortexing and sonicating.Nanopure water (400 μl) was added to each sample and returned to theincubator for 10 minutes. After sonicating for 1 minute, the layers wereseparated by centrifuging at 15,300×g for 3 minutes. The butanol layerswas transferred to a new tube and dried down using a Savant (Holbrook,N.Y.) SpeedVac Plus. Dried samples were completely resuspended inmethanol (375 μl) and agitated in a bath sonicator for 2 minutes. Theextracts were then derivatized with O-phthalaldehyde (OPA) (MolecularProbes, Eugene, Oreg.). In brief, 50 mg of OPA were dissolved in ethanol(1.0 mL). Into 0.5 M boric acid (24.75 mL), 0.25 mL of the OPA inethanol was added and mixed. Finally, 2-mercaptoethanol (13 μl) wasadded and mixed to make up the OPA derivatization solution. To eachsample 10 mM disodium EDTA (50 μl) was added followed by 0.5 M boricacid (50 μl) and OPA solution (25 μl). Samples were incubated at roomtemperature protected from light for 20 minutes.

[0288] The HPLC analysis was performed using a Beckman (Fullerton,Calif.) System Gold 118 Solvent Module and 507e Autosampler. A Jasco(Easton, Md.) FP-920 Intelligent Fluorescence Detector was used with anexcitation wavelength of 330 nm and emission wavelength of 455 nm. Thederivatized samples (50 μl injection) were separated on a Beckman(Fullerton, Calif.) Ultrasphere ODS 4.6 mm×25 cm column with a 1.5 cmPerkin Elmer (Norwalk, Conn.) NewGuard RP-18 guard column. The solventsystem was methanol, glacial acetic acid, 1 M tetrabutlyammoniumdihydrogen phosphate, Nanopure water (82.9:1.5:0.9:14.7, v/v) run at 1.5mL/minute. Chromatograms were analyzed using the Beckman System GoldNouveau software. Results are shown in Table 1.

[0289] The only sphingolipid base that accumulated in cardiomyocytes inresponse to hypoxia was sphingosine (SPH). Levels of S-1-P and SPC werenot increased by hypoxia. Cumulative data from several experimentsdemonstrated that hypoxia produces a 6.4-fold increase in SPHproduction. The increase in total cell SPH is not reflected in anincreased intracellular content. Instead, the majority of the SPHproduced in response to hypoxia is released from the cells into thecell-conditioned media. These data demonstrate a 18-fold increase in theextracellular SPH content of hypoxic cardiomyocytes.

[0290] The short time (5 hrs) of hypoxia employed did not result inappreciable necrotic or apoptotic cell death but was associated withsignificant TNFα release. Pretreatment with the TNFRII:Fc receptorfragment (Mohler et al., J. Immunol. 151:1548-1561, 1993), resulted inthe significant (p<0.001) reduction (˜3-fold) of the SPH release. TNFαreceptor fragment pretreatment did not mitigate SPH-triggered apoptosis(SPH only, no TNF), indicating that SPH production is a step in thesignal cascade that is “downstream” from TNFα binding to its receptors.

Example 4

[0291] Blood SPH is Converted to S-1-P

[0292] Studies with human blood obtained from normal subjects suggestthat blood platelets are capable of converting SPH to S-1-P because oftheir rich source of sphingosine kinase (Yatomi et al., J. Biochem.121:969-973, 1997; Yatomi et al., J. Biol. Chem. 272:5291-5297, 1997;Yatomi et al., Blood 86:193-202, 1995). In such experiments,commercially supplied SPH was added to blood serum which, in the absenceof cellular components, was found to be unable to convert SPH to S-1-P.Without wishing to be bound by any particular theory, applicants believethat the ischemic heart is the major source of serum SPH and thatcardiac-derived SPH could be converted to S-1-P by blood platelets (U.S.Pat. No. 6,210,976 B1, and published PCT patent application WO98/57179).

[0293] Experiments were carried out to determine the fate of any SPHthat might be released from cells or platelets into the extracellularcompartment. Whole blood samples were incubated in vitro for up to 15hours with ³H-SPH, followed by thin layer chromatography (TLC) toexamine which, if any, of the known metabolic products of thesphingomyelin pathway were radiolabeled as a result of metabolism ofradiolabeled SPH.

[0294] The results (FIG. 3) indicate that, in whole blood, theradiolabeled SPH was not converted to ceramide or sphingomyelin, nor wasit metabolized to dimethylsphingosine or dihydrosphingosine (see FIG.2). The major metabolite of the pathway that was appreciably labeled wassphingosine-1-phosphate (S-1-P), presumably due to the action of thevery active sphingosine kinase present in blood platelets (Yatomi etal., J. Biochem. 121:969-973, 1997; Yatomi et al., J. Biol. Chem.272:5291-5297, 1997; Yatomi et al., Blood 86:193-202, 1995).

[0295] A substantial amount of the time-dependent conversion of ³H-SPHto ³H-S-1-P occurred within 20 minutes and represented a conversion of86%. TABLE 1 Sphingolipid Production in Cultured Cardiomyocytes DuringNormoxia, Hypoxia and Reoxygenation Hypoxia 12 hours for neonates 7hours Reoxygenation Normoxia for adults 5 hrs Neonatal cardiomyocytes %Permeabilized cells 1.7 ± 1.3 3.15 ± 1.3  7.45 ± 2.5  % Apoptotic cells1.17 ± 0.37 4.58 ± 0.74 *23.5 ± 2.7  TNFα (pg/106 cells)  561 ± 13.4*979 ± 26.7  — SPH (pmol/106 cells)  5.8 ± 0.43 *77.6 ± 6.24  — Adultcardiomyocytes % Permeabilized cells 4.71 ± 1.18 9.5 ± 2.9 — % Apoptoticcells 8.12 ± 1.76 *26.7 ± 2.9  — TNFα (pg/106 cells) 810 ± 155 *8660 ±3150  — SPH (pmol/106 cells) 238 ± 39  *3860 ± 547  —

[0296] The data also demonstrate that S-1-P was very stable in wholeblood for the 15-hour time course of the experiment. Only after severalhours was it evident that a small but measurable amount oft-2-hexadecanal (a.k.a. palmitaldehyde) was produced as a consequence ofthe low level of S-1-P lyase present in platelets that can convert S-1-Pto hexadecanal and ethanolamine phosphate (Yatomi et al, J. Biochem.121:969-973, 1997; Yatomi et al., J. Biol. Chem. 272:5291-5297, 1997;Yatomi et al., Blood 86:193-202, 1995).

[0297] It is possible that blood platelets were the major reservoir ofSPH-derived S-1-P (Yatomi et al, J. Biochem. 121:969-973, 1997). Anotherpossible reservoir of S-1-P could be heart cells. Some studies haveindicated that SPH is produced intracellularly by culturedcardiomyocytes, although these studies did not present evidence of S-1-Prelease/secretion (U.S. Pat. No. 6,210,976 B1 and Published PCTApplication WO 98/57179).

Example 5

[0298] Blood S-1-P is Cardiotoxic and Dependent on S-1-P Receptors

[0299] During ischemia-induced myocardial infarction, several importantevents occur as a consequence of the pathophysiology. In 74% of thecases of AMI, the sudden cardiac death is associated with significantthrombus (blood clot) in the coronary artery supplying blood to theinfarcted region of the myocardium (Davies et al., N. Engl. J. Med.310:1137-1140, 1984). “Infarcted” areas are those in which cells aredead or have sustained so much damage that they are fated to die. Thereis a profound negative inotropic effect (i.e., loss of contractility) onthe myocardial cells induced by the ischemia itself. Intracellularcalcium control is deregulated with diastolic (resting) calcium steadilyrising in an uncontrolled fashion accompanied by a decrease in systolic(contractile) calcium leading to an eventual ‘calcium overload’.

[0300] Without wishing to bound by any particular theory, applicantsbelieve that these effects are a consequence of the initial SPHproduction by pre-AMI and post-AMI ischemic cardiac cells and thesubsequent production of S-1-P by nearby platelets. It is also believedthat S-1-P activates nearby platelets through calcium deregulation ofthe platelets and that platelet-derived S-1-P is released into theserum.

[0301] Exogenously applied S-1-P is capable of activating bloodplatelets (Yatomi et al., J. Biol. Chem. 272:5291-5297, 1997; Yatomi etal., Blood 86:193-202, 1995). It has been suggested that the increasedserum S-1-P has two major actions, both of which are cardiotoxic. Thefirst is to act on the platelets to promote the clotting response andfurther exacerbate the ischemia. Secondly, the platelet-derived S-1-Pacts in a paracrine fashion on neighboring endothelial and myocardialcells to promote calcium deregulation and apoptosis. On thecardiomyocytes, the consequence is to promote calcium overload. Theendothelial cell response is to promote vasoconstriction and furtherlimit the blood supply through the coronary vasculature. It hasdemonstrated that S-1-P applied to cultured cardiomyocytes resulted inapoptosis and dramatic increases in diastolic calcium followed inminutes by decreases in systolic calcium and eventual calcium overloadreminiscent of what happens to the ischemic myocardium. It has alsodemonstrated that both rat and human cardiac tissue express genes forthe recently identified S-1-P receptors of the EDG (endothelialdifferentiation gene) family, and that these receptors mediate thecalcium deregulation (Nakajima et al., Biophysical J. 78:319 A, 2000).

[0302] Extracellular S-1-P is the ligand for a novel class of Gprotein-coupled receptors (GPCRs). Such receptors were first describedas an orphan GPCRs cloned from human umbilical vein endothelial cells(Hla et al., J. Biol. Chem. 265:9308-9313, 1990; Lynch et al., TrendsPharmacol. Sci. 20:473-475, 1999). Tissue expression of the EDG-1/3/5genes in the murine system indicates that heart and lung have thehighest overall expression of these three genes (Zhang et al., Gene227:89-99, 1999). However, cardiac tissues are composed of diverse celltypes, including endothelial cells, which have high levels of EDG-1expression (Hla et al., J. Biol. Chem. 265:9308-9313, 1990). AlthoughEDG-1/3/5 are expressed in C2C12 skeletal muscle myoblasts (Meacci etal, FEBS Letters 457:184-188, 1999), cardiomyocyte-specific expressionof the EDG genes has not been elucidated. Recently a EDG-1 gene and itsprotein expression were determined in primary cultures of neonatal ratcardiomyocytes and in adult rat ventricular tissue (Nakajima et al.,Biophysical J. 78:319 A, 2000). Cultured cardiomyocytes were used toevaluate the functional role of EDG receptors, including the ability ofS-1-P to modulate intracellular calcium levels.

[0303] Importantly, the calcium deregulatory responses evoked by S-1-Pin cultured cardiomyocytes are quite similar to the calcium deregulationseen in models of acute myocardial infarction (Lee et al., Circ.78:1047-1059, 1988; Smith et al., Amer. Heart J. 103:716-723, 1982;Kihara et al, Circ. Res. 65:1029-1044, 1989). These characteristicresponses include: increases in diastolic calcium (and calciumoverload), decreases in systolic calcium (the negative inotropic state)and the production of abnormal oscillatory beating behavior(arrhythmias) and cessation of activity, all of which were seen in thecardiomyocytes when treated with S-1-P. Additionally, the averagecalcium level combining both diastolic and systolic responses issignificantly increased by S-1-P treatment. Taken together, these datasuggest that S-1-P, possibly acting via EDGRs, increases the influx ofextracellular calcium which then causes the calcium overload.

[0304] It is well known that calcium deregulation is a prerequisite toapoptosis (Nagnelli et al., Bioch. Biophys. Res. Comm. 204:84-90, 1994).Previously published data indicate that rat cardiomyocytes in cultureundergo apoptotic cell death when exposed to sphingosine (Krown et al,J. Clin. Invest. 98:2854-2865, 1996). It is also suggested that theS-1-P present in putative high levels in cardiac circulation could acton cardiac cell S-1-P receptors to produce profound negative inotropiceffects and cell death by apoptosis.

[0305] Thus, it is likely that the pre-AMI ischemic myocardium producesthe initial instruments of its own destruction, namely, variouscytokines and, importantly, SPH. The secreted SPH, acting indirectlythrough platelet-derived S-1-P, promotes the activation of S-1-Preceptors on platelets, endothelial cells and cardiomyocytes. Bloodclotting, vasoconstriction and myocardial calcium overload are theconsequences with myocardial infarction as the ultimate result. As thegrowing ischemia and the area of infarction challenges more cardiactissue, additional SPH is released and a positive feedback loop resultsuntil substantial cell death occurs.

[0306] Although not wishing to be bound to any particular theory, it ispossible that during low levels of ischemia and cardiac stress (e.g.,hyptertension), the cardiac cells produce cytokines and sphingolipids asextracellular signaling molecules that serve to precondition the heartto these stresses. Protection can come from preconditioning the heartitself via protection against calcium overload, cell death andarrhymthias or by producing a hypernatating myocardial state to lowerenergy demands during ischemia.

Example 6

[0307] Use of Antibodies in Sphingolipid-Based Therapy

[0308] This Example describes how sphingolipid-based cardiovasculartherapy can be realized by the use of antibodies and derivatives thereof(single-chain Fv's, CDR's, etc.) that specifically bind certainmolecules as therapeutic agents. Such antibodies are directed to, by wayof non-limiting example, antibodies to sphingolipids and receptorsthereof.

[0309] Antibodies to Sphingolipid Receptors

[0310] One type of therapeutic antibody specifically sphingolipidreceptors that carry out the cellular internalization of undesirablesphingolipids. In some cases, the delivery into the cell of anundesirable sphingolipid results in a sequence of events having anundesirable effect. Antibodies to such receptors prevent the entry ofthe undesirable sphingolipid into cells, thus avoiding the undesirableconsequences of such entry. For example, the undesirable, toxic and/orcardiotoxic sphingolipid S-1-P has many actions that are dependent uponbinding to sphingolipid receptors, including without limitation Edgreceptors (Example 15). Antibodies to receptors that block the bindingof a undesirable, toxic and/or cardiotoxic sphingolipid are developedand tested for their ability to inhibit the binding of S-1-P to itsreceptors, as well as for their ability to block post-binding eventsthat lead to cardiotoxic effects. Antibodies to Edg receptors are known,and are in some instances commercially available. For example,antibodies to Edg-1, -7 and -8 are available from Oncogene ResearchProducts; antibodies to Edg-2 are available from Calbiochem; antibodiesto Edg-4 are available from Antibody Solutions (Palo Alto, Calif.); andantibodies to Edg-5 are available from Exalpha Biologicals, Inc.(Boston, Mass.).

[0311] Antibodies to Sphingolipids

[0312] One type of therapeutic antibody specifically binds undesirablesphingolipids. Such antibodies bind sphingolipids in order to achievebeneficial effects such as, e.g., (1) lowering the effectiveconcentration of available (i.e., unbound) undesirable, toxic and/orcardiotoxic sphingolipids (and/or the concentration of their metabolicprecursors) that would otherwise be free to exert their harmful effectson cells (including, by way of non-limiting example, removingundesirable, toxic and/or cardiotoxic sphingolipids and their metabolicprecursors from blood via ex vivo treatments); (2) to inhibit thebinding of an undesirable, toxic and/or cardiotoxic sphingolipid to acellular receptor therefor, and/or to lower the concentration of asphingolipid that is available for binding to such a receptor; and/or(3) preventing the metabolic conversion of a first sphingolipid into asecond and more undesirable, toxic and/or cardiotoxic sphingolipid,and/or to lower the concentration of such a precursor that is availablefor enzymatic conversion into a undesirable, toxic and/or cardiotoxicsphingolipid.

[0313] Examples of such therapeutic effects include but are not limitedto the use of (i) anti-S-1-P antibodies to lower the concentration ofavailable S-1-P, thereby blocking or at least limiting S-1-P'scardiotoxic and thrombogenic effects, and/or (ii) anti-SPH antibodies toprevent the metabolic conversion of SPH to the more undesirable, toxicand/or cardiotoxic sphingolipid S-1-P.

[0314] To produce mAb to phospholipids, acid-treated SalmonellaMinnesota are administered directly into a mouse spleen using protocolsessentially according to the methods of Umeda et al. that have been usedto make mAbs to phosphatidylserine (J. Immunol. 143:2273-2279, 1989; seealso Reza et al., FEBS Lett. 339:229-233, 1994).

[0315] For production of anti-SPH antibodies, the acid-treated S.Minnesota are coated with SPH and injected into the mouse spleen priorto cell fusion to produce a hybridoma that secretes anti-SPH mAb.Similar methods are used to produce anti-S-1-P mAb and anti-SPC mAb.

[0316] Additionally or alternatively, fatty acid free BSA-sphingolipidconjugates are used as the immunogen in order to present unique epitopesto the animal. Appropriate steps are taken to ensure that the mAbsproduced in this fashion are directed to sphingolipids of choice and notto oxidized lipid or protein-lipid adducts (Horkko et al., J. Clin.Invest. 98:815-825, 1996; Palinski et al., J. Clin. Invest. 98:800-814,1996).

[0317] Antibodies to S-1-P

[0318] In order to develop antibodies to S-1-P, guinea pigs wereimmunized IP once a week for 4 weeks with 1 mg of KLH-derivatizedsphingolipid. The protocols that were used are essentially those ofHorkko et al. (J. Clin. Invest. 98:815-825, 1996) and Palinski et al.(J. Clin. Invest. 98:800-814, 1996).

[0319] In brief, the animals were given weekly injections over a periodof several weeks. In the first week, 150 ug of immunogen in CompleteFreund's adjuvant was injected. During the second, third, fourth, fifthand sixth weeks, 100 ug of immunogen in Incomplete Freund's adjuvant wasinjected into the guinea pigs. Serum samples collected from theimmunized guinea pigs were shown to contain antibodies to S-1-P by useof an ELISA assay. The ELISA was carried out essentially according tothe procedures described by Horkko et al. (J. Clin. Invest. 98:815-825,1996) and Palinski et al. (J. Clin. Invest. 98:800-814, 1996). The serumsamples had a titer of 140,000 Relative Lumiscent Units/100 ms.

Example 7

[0320] Modulation of Sphingosine-1-Phosphate (S-1-P) Metabolism

[0321] The concentration of the undesirable, toxic and/or cardiotoxicsphingolipid S-1-P is lowered (i) by stimulating reactions that utilizeS-1-P as a reactant (i.e., reactions that degrade S-1-P, e.g., Rxns. #1and #2 in FIG. 2) and, additionally or alternatively, (ii) by inhibitingchemical reactions that yield S-1-P as a product (i.e., reactions thatproduce S-1-P, e.g. Rxn. #3 in FIG. 2). Such stimulation and/orinhibition is achieved by, for example, (1) increasing the amount of,and/or enhancing the activity of, enzymes that catalyze the catabolism(degradation) of S-1-P and, additionally or alternatively, (2) reducingthe amount of, and/or or inhibiting or completely blocking the activityof, enzymes that catalyze the anabolism (production) of S-1-P.

[0322] In instances where the goal is to increase the concentrationenzymes that degrade S-1-P, pharmaceutical formulations of such enzymesare administered to a patient. S-1-P-degrading enzymes are purified froma variety of mammals and other animals, or produced in vitro from cellsusing recombinant DNA techniques.

[0323] Inhibition of Production of S-1-P

[0324] The inhibition of enzymes that catalyze reactions that yieldS-1-P (i.e., reactions that have S-1-P as a product) is expected toresult in the reduction or complete inhibition of the production ofS-1-P. Such enzymes include but are not limited to the following:

[0325] Sphingosine Kinase (SPH kinase) catalyzes the conversion of SPHto S-1-P (Rxn. #3 in FIG. 2; see also FIG. 1). A genetic sequenceencoding human SPH-kinase has been described (Melendez et al., Gene251:19-26, 2000). Three human homologs of SPH kinase (SKA, SKB and SKC)have been described (published PCT patent application WO 00/52173).Murine SPH kinase has also been described (Kohama et al., J. Biol. Chem.273:23722-23728, 1998; and published (PCT patent application WO99/61581). Published PCT patent application WO 99/61581 to Spiegel isstated to describe nucleic acids encoding a sphingosine kinase.Published PCT patent application WO 00/52173 to Munroe et al. is statedto describe nucleic acids encoding homologues of sphingosine kinase.Other SPH Kinases are described by Pitson et al., “Human sphingosinekinase: purification, molecular cloning and characterization of thenative and recombinant enzymes”, Biochem J. 350:429-441, 2000; andpublished PCT application WO 00/70028 to Pitson et al.; and Liu et al.,“Molecular Cloning and Functional Characterization of a Novel MammalianSphingosine Kinase Type 2 Isoform”, The Journal of Biological Chemistry,275:19513-19520, 2000; Vadas et al., “Sphingosine Kinase and UsesThereof”, PCT/AU01/00539, published as WO 01/85953 on Nov. 15, 2001;Rastelli, “Novel Sphingosine Kinases”, PCT/US01/04789, published as WO01/60990 on Aug. 23, 2001; Allen et al., “Human Sphingosine KinaseGene”, PCT/EP00/09498, published as WO 01/31029 on May 3, 2001.

[0326] Inhibitors of SPH kinase include but are not limited toN,N-dimethylsphingosine (DMS) (Edsall et al., Biochem. 37:12892-12898,1998); D-threo-dihydrosphingosine (Olivera et al., Nature 365:557-560,1993); and Sphingoid bases (Jonghe et al., “Structure-ActivityRelationship of Short-Chain Sphingoid Bases As Inhibitors of SphingosineKinase”, Bioorganic & Medicinal Chemistry Letters 9:3175-3180, 1999)Assays of SPH kinase useful for evaluating these and other known orpotential SPH kinase inhibitors include those disclosed by Olivera etal., “Assaying Sphingosine Kinase Activity”, Methods in Enzymology,311:215-223, 1999; Caligan et al., “A High-Performance LiquidChromatographic Method to Measure Sphingosine 1-Phosphate and RelatedCompounds from Sphingosine Kinase Assays and Other Biological Samples”,Analytical Biochemistry, 281:36-44, 2000.

[0327] Pharmaceutical compositions of these and other inhibitors of SPHkinase, especially those that are formulated for rapid cardiac delivery,are used for this form of sphingolipid-based cardiovascular therapy.

[0328] Inhibition of SPH kinase may lead to an accumulation of itssubstrate, SPH, which is also an undesirable sphingolipid, albeitgenerally less harmful than S-1-P. In order to avoid or mitigate thiseffect should it occur, additional agents are concurrently administeredto (i) stimulate an enzyme that has SPH as a substrate, with the provisothat the enzyme should not be one that has S-1-P as a product (such as,e.g., ceramide synthase; see below); and, additionally or alternatively,(ii) inhibit an enzyme that has SPH as a product.

[0329] Stimulation of Destruction of S-1-P

[0330] The stimulation of enzymes that catalyze reactions that degradeS-1-P (i.e., reactions that have S-1-P as a reactant) is expected toresult in the stimulation of degradation of S-1-P molecules. Suchenzymes include but are not limited to the following:

[0331] S-1-P Lyase catalyzes the conversion of S-1-P to ethanolamine-Pand (a.k.a. t-2-hexadecanal) palmitaldehyde (Veldhoven et al., Adv.Lipid Res. 26:67-97, 1993; Van Veldhoven, “Sphingosine-1-phosphateLyase” Methods in Enzymology, 311:244-254, 1999; Rxn. #1 in FIG. 2).Yeast (Lanterman et al., Biochem. J. 332:525-531, 1998), murine (Zhou etal., Biochem. Biophys. Res. Comm. 242:502-507, 1998) and human(published PCT patent applications WO 99/38983 and WO 99/16888) S-1-Plyase genes have been described. Published PCT patent application WO99/16888 to Saba et al. is stated to describe S-1-P lyase DNA andprotein sequences. U.S. Pat. No. 6,187,562, Published PCT patentapplication WO 99/38983 to Duckworth et al. is stated to describe aS-1-P lyase. See also Van Veldhoven et al., “Humansphingosine-1-phosphate lyase: cDNA cloning, functional expressionstudies and mapping to chromosome 10q22¹”, Biochimica et Biophysica Acta1487:128-134, 20000); and. Mandala et al., “Molecular cloning andcharacterization of a lipid phosphohydrolase that degradessphingosine-1-phosphate and induces cell death”, PNAS, 97:7859-7864,2000.

[0332] Pharmaceutical compositions of agents that are stimulators ofS-1-P lyase, especially those that are formulated for rapid cardiacdelivery, are used for this form of sphingolipid-based cardiovasculartherapy.

[0333] S-1-P Phosphatase (a.k.a. SPP phosphohydrolase) is a mammalianenzyme that catalyzes the conversion of S-1-P to sphingosine (Rxn. #2 inFIG. 2) (Mandala et al., Proc. Nat. Acad. Sci. 95:150-155, 1998; Mandalaet al., Proc. Nat. Acad. Sci. 97:7859-7864, 2000; Mandala,“Sphingosine-1-Phosphate Phosphatases”, Prostaglandins & other LipidMediators, 64:143-156, 2001; Brindley et al., “Analysis of Ceramide1-phosphate and Sphingosine-1-phosphate Phosphatase Activities”, Methodsin Enzymology, 311:233-244, 1999). Two S-1-P phosphatases, LBP1 andLBP2, have been isolated from yeast (Mandala et al., J Biol. Chem.272:32709-32714, 1997). Mandala et al., “MammalianSphingosine-1-Phosphate Phosphatase”, PCT/UW01/03879, published asWO01/57057 on Aug. 9, 2001.

[0334] Pharmaceutical compositions of agents that are stimulators ofS-1-P phosphatase, especially those that are formulated for rapidcardiac delivery, are used for this form of sphingolipid-basedcardiovascular therapy.

Example 8

[0335] Modulation of Sphingosine (SPH) Metabolism

[0336] The concentration of the undesirable, toxic and/or cardiotoxicsphingolipid SPH is lowered (i) by stimulating reactions that utilizeSPH as a reactant (i.e., reactions that degrade SPH without producingS-1-P) and, additionally or alternatively, (ii) by inhibiting chemicalreactions that yield SPH as a product (i.e., reactions that produce SPH,e.g., Rxn. #5 in FIG. 2). Such stimulation and/or inhibition is achievedby, for example, (1) increasing the amount of, and/or enhancing theactivity of, enzymes that catalyze the catabolism (degradation) of SPHand, additionally or alternatively, (2) reducing the amount of, and/oror inhibiting or completely blocking the activity of, enzymes thatcatalyze the anabolism (production) of SPH. Because SPH is convertedinto S-1-P by enzymes such as SPH kinase (Rxn. #3 in FIG. 2), loweringthe concentration of the undesirable, toxic and/or cardiotoxicsphingolipid SPH is not only therapeutic in its own right but, if donewithout converting SPH to S-1-P, has the additional therapeutic benefitof lowering the production of the more undesirable, toxic and/orcardiotoxic sphingolipid S-1-P.

[0337] In instances where the goal is to increase the concentration ofenzymes that degrade SPH, pharmaceutical formulations of such enzymesare administered to a patient. SPH-degrading enzymes, such as SPH Kinaseand ceramide synthase (Rxns. #3 and 4, respectively, in FIG. 2) arepurified from a variety of mammals, including humans, and other animals;or are produced in vitro using, e.g., recombinant DNA techniques.

[0338] Inhibition of Production of SPH

[0339] The inhibition of enzymes that catalyze reactions that yield SPH(i.e., reactions that have SPH as a product) is expected to result inthe reduction or complete inhibition of the production of SPH. Suchenzymes include but are not limited to the following:

[0340] Ceramidase (CDase) catalyzes the conversion of ceramide to SPH(Rxn. #5 in FIG. 2; see also FIG. 1). For a review, seeNikolova-Karakashian et al., “Ceramidases”, Methods in Enzymology,311:194-201, 1999. At least two types of ceramidases are known in theart, ceramidase I and ceramidase II, which differ in terms of pH optima(Sugita et al., Biochim. Biophys. Acta. 398:125-131, 1975; Yada et al.,J. Biol. Chem. 270:12677-12684, 1995). Ceramidases are disclosed by Taniet al., “Purification and Characterization of a Neutral Ceramidase fromMouse Liver: A Single Protein Catalyzes the Reversible Reaction in WhichCeramide is Both Hydrolyzed and Synthesized”, The Journal of BiologicalChemistry 275:3462-3468, 2000; Mao et al., “Cloning and Characterizationof a Saccharomyces cerevisiae Alkaline Ceramidase with Specificity forDihydroceramide”, The Journal of Biological Chemistry 275:31369-31378,2000; Zhang et al., “Human Acid Ceramidase Gene: Novel Mutations inFarber Disease”, Molecular Geneetics and Metabolism 70:301-309, 2000;Mao et al., “Cloning of an Alkaline Ceramidase from Saccharomycescerevisiae: An Enzyme with Reverse (CoA-Independent) Ceramide SynthaseActivity”, The Journal of Biological Chemistry 275:6876-6884, 2000;Okino et al., “Molecular Cloning, Sequencing, and Expression of the GeneEncoding Alkaline Ceramidase from Pseudomonas aeruginosa: Cloning of ACeramidase Homologue from Mycobacterium Tuberculosis”, 274:36616-36622,1999; Kita et al., “Reverse hydrolysis reaction of a recombinantalkaline ceramidade of Pseudomonas aeruginosa” Biochimica et BiophysicaActa 1485:111-120, 2000; Li et al., “The Human Acid Ceramidase Genes(ASAH): Structure, Chromosomal Location, Mutation Analysis, andExpression”, Genomics, 62:223-231, 1999; Mao et al., “Cloning andCharacterization of a Novel Human Alkaline Ceramidase: A MammalianEnzyme That Hydrolyzes Phytoceramide”, The Journal of BiologicalChemistry, 276:26577-26588, 2001; Mitsutake et al., “Purification,Characterization, Molecular Cloning, and Subcellular Distribution ofNeutral Ceramidase of Rat Kidney”, The Journal of Biological Chemistry,276:26249-26259, 2001; Ito et al., “Ceramidase Gene”, PCT/JP00/01802,published as WO00/58448 on Oct. 5, 2000; Okino et al., “MolecularCloning, Sequencing, and Expression of the Gene Encoding AlkalineCeramidase from Pseudomonas aeruginosa”, The Journal of BiologicalChemistry, Vol. 274, Dec. 17, 1999, pp. 36616-36622; and Bawab et al.,“Molecular Cloning and Characterization of a Human MitochondrialCeramidase”, The Journal of Biological Chemistry, 275:21508-21513, 2000

[0341] Inhibitors of ceramidase include but are not limited toD-erythro-MAPP and L-erythro-MAPP (Bielawska et al., J. Biol. Chem.271:12646-12654, 1996; Hannun et al., “Inhibitor of Ceramidase”,PCT/US96/17769, published as WO97/44019 on Nov. 27, 1997); andN-oleoyl-ethanolamine (NOE; Sugita et al., 1975; Yada et al., J. Biol.Chem. 270:12677-12684, 1995; Meroni et al., “Effect ofN-Acetylsphingosine (C2) and the Ceramidase Inhibitor(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol on theRegulation of Sertoli Cell Function”, Journal of Andrology, 20:619-625,1999). As CDase is inhibited by endogenous sphingolipids such as SPH andSM (Hise et al., J. Clin. Invest. 77:768-773, 1986); nontoxic syntheticsphingolipids that inhibit CDase are also used. See also Hannun et al.,“Inhibitors of Ceramidase”, U.S. Pat. No. 5,851,782, issued Dec. 22,1998.

[0342] Methods for assaying ceramidase activity are disclosed by He etal.; “A Fluorescence-Based High-Performance Liquid Chromatography Assayto Determine Acid Ceramidase Activity”, Analytical Biochemistry,274:264-269, 1999. Pharmaceutical compositions of these and otherinhibitors of CDase, especially those that are formulated for rapidcardiac delivery, are used for this form sphingolipid-basedcardiovascular therapy.

[0343] S-1-P Phosphatase (a.k.a. SPP phosphohydrolase) catalyzes theconversion of S-1-P to sphingosine (Rxn. #2 in FIG. 2) and is describedin more detailed in the preceding Example 8. Inhibition of S-1-Pphosphatase has the beneficial result of lowering SPH production;however, inhibition of S-1-P phosphatase potentially includes theundesirable effect of inhibiting the degradation of S-1-P. It is thususeful to include one or more stimulators of an enzyme that degradesS-1-P (such as, e.g., S-1-P lyase; see above), in pharmaceuticalcompositions used for this form sphingolipid-based cardiovasculartherapy.

[0344] Stimulation of Destruction of SPH

[0345] The stimulation of enzymes that catalyze reactions that degradeSPH (i.e., reactions that have SPH as a reactant, e.g., Rxns. #3 and #4in FIG. 2) is expected to result in the stimulation of degradation ofSPH molecules. In general, it is preferable that such reactions do notyield a undesirable, toxic and/or cardiotoxic sphingolipid, especiallyS-1-P, as a product (e.g., Rxn. #4 in FIG. 2, which is catalyzed byceramide synthase). SPH kinase may be stimulated to enhance thedegradation of SPH but the reaction it catalyzes produces theundesirable sphingolipid S-1-P; accordingly, stimulators of SPH kinaseare preferably combined with stimulators of enzymes that degrade S-1-P,e.g., S-1-P lyase (Rxn. # 1 in FIG. 2).

Example 9

[0346] Modulation of Ceramide (CER) Metabolism

[0347] The concentration of CER is lowered (i) by stimulating reactionsthat utilize CER as a reactant (i.e., reactions that degrade CER),preferably those reactions that do not yield SPH as a product (e.g.,Rxn. # 5 in FIG. 2); and, additionally or alternatively, (ii) byinhibiting chemical reactions that yield CER as a product (i.e.,reactions that produce CER), preferably those reactions that do not useSPH as a reactant (e.g., Rxn. #4 in FIG. 2). Such stimulation and/orinhibition is achieved by, for example, (1) increasing the amount of,and/or enhancing the activity of, enzymes that catalyze the catabolism(degradation) of CER and, additionally or alternatively, (2) reducingthe amount of, and/or or inhibiting or completely blocking the activityof, enzymes that catalyze the anabolism (production) of CER. However,such CER-producing enzymes do not use SPH as a substrate, as inhibitionof such enzymes (e.g., ceramide synthase) is expected to result in anincrease in the level of SPH. Because CER is directly converted into themore undesirable, toxic and/or cardiotoxic sphingolipid SPH by enzymessuch as ceramidase (Rxn. # 5 in FIG. 2), lowering the concentration ofCER has the therapeutic benefit of lowering the production of the moreundesirable, toxic and/or cardiotoxic sphingolipid SPH; this effectwhich results in a lowered production of S-1-P by SPH kinase.

[0348] In instances where the goal is to increase the concentration ofenzymes that degrade SPH, pharmaceutical formulations of such enzymesare administered to a patient. SPH-degrading enzymes are purified from avariety of mammals, including humans, and other animals; or produced invitro from cells using recombinant DNA techniques.

[0349] Inhibition of Production of CER

[0350] The inhibition of enzymes that catalyze reactions that yield CER(i.e., reactions that have CER as a product) is expected to result inthe reduction or complete inhibition of the production of CER. Suchenzymes include but are not limited to the following:

[0351] Ceramide Synthase (CER synthase), also known as sphingosineN-acyltransferase, catalyzes the acetylation of dihydrosphingosine (Rxn.#10 in FIG. 2) which leads to the production of ceramide.

[0352] Inhibitors of CER synthase include the fungal toxin Fumonisin B1(Merrill et al., J. Lipid Res. 26:215-234A, 1993; Wang et al., Adv.Lipid Res. 26:215-234, 1993; Lee et al., Biochem. J. 334:457-461, 1998;Xu et al., J. Biol. Chem. 273:16521-16526, 1998; Lochhead et al, KidneyInt. 54:373-381, 1998; Tsunoda et al., J. Biochem. Mol Toxicol.12:281-289, 1998); derivatives of fumonisin (Humpf et al, J. Biol. Chem.273:19060-19064, 1998); alternaria toxins (Id. and Mandala et al., J.Antibiot. 48:349-356, 1995); viridiofungins (Merrill et al., J. LipidRes. 26:215-234A, 1993; astralifungins (Mandala et al., J. Antiobiot.48:349-356, 1995; Furneisen et al., Biochim. Biophys. Acta. 1484:71-82,2000); and D-erythro-N-myristoyl 2-amino-1-phenylpropanol (Hunnan,Science 274:1855-1859, 1996). Pharmaceutical compositions of these andother inhibitors of CER synthase, especially those that are formulatedfor rapid cardiac delivery, are used for this form sphingolipid-basedcardiovascular therapy.

[0353] Ceramide-1-P Phosphatase (CER-1-P phosphatase) catalyzes theproduction of ceramide from ceramide 1-P (Rxn. #17 in FIG. 2). SeeShinghal et al., “Ceramide 1-Phosphate Phosphatase Activity in Brain”,Journal of Neurochemistry, 61:2279-2285, 1993; Boudker et al.,“Detection and Characterization of Ceramide-1-phosphate PhosphataseActivity in Rat Liver Plasma Membrane”, The Journal of BiologicalChemistry, 268:22150-22155, 1993.

[0354] Inhibitors of CER-1-P phosphatase include but are not limited tomanganese; cobalt; NaF; propranolol; phenylglyoxal; and n-ethylmaleimide(Fureisen et al, Biochim. Biophys. Acta. 1484:71-82, 2000).Pharmaceutical compositions of these and other inhibitors of CER-1-Pphosphatase, especially those that are formulated for rapid cardiacdelivery, are used for this form of sphingolipid-based cardiovasculartherapy.

[0355] Sphingomyelinase (SMase) catalyzes the conversion ofsphingomyelin to ceramide (Rxn. #7 in FIG. 2; see also FIG. 1). Variousisoforms of SMase have been described. These include nSMases (n forneutral pH isoform), aSMases (a for acidic pH isoform), and alkalineSMases including an SMase isoform found in the gut. Both the acidic andneutral forms of SMase are endogenous to cardiac tissue (Andrieu-Abadieet al, FASEB J. 13:1501-1510, 1999), and a novel form of a high turnoversphingomyelinase localized in the junctional T-tubule membranes (Ghoshet al., Mol. Cellular Biochem. 189:161-168, 1998). The neutral form ofSMase is exposed to the extracellular surface of the membrane (Mohan etal., Biochem Biophys Acta 777:339-342, 1984) and would thus beaccessible to lipid-insoluble agents. SMases are described in greaterdetail in Example 13.

[0356] It has been demonstrated that the sphingomyelinase inhibitor,L-carnitine, blocks doxorubicin-induced apoptosis coincident with theinhibition of ceramide production (Andrieu-Abadie et al, FASEB J.13:1501-1510, 1999; Katircioglu, et al., J. Cardiovasc. Surg. 41:45-50,1999; Gunther, Eur. J. Pharma. 406:123-126, 2000.

[0357] Inhibitors of SMase include but are not limited to gentamicin(Ghosh et al., J. Biol. Chem. 262:12550-12556, 1987) and gentamicinderivatives, and other aminoglycosides, as is described in more detailelsewhere herein.

[0358] Several inhibitors of SMase have been prepared that are based onthe structures of the naturally occuring compounds Scyphostatin andManumycin. In addition to naturally occuring compounds such asScyphostatin and Manumycins A-D, several inhibtors have been synthesizedin vitro. These include without limitation those described by Arenz etal., “Synthesis and Biochemical Investigation of Scyphostatin Analoguesas Inhibitors of Neutral Sphingomyelinase”, Bioorganic & MedicinalChemistry, 9:2901-2904, 2001; Arenz et al., “Synthesis of the FirstSelective Irreversible Inhibitor of Neutral Sphingomyelinase”, Eur. J.Org. Chem., 137-140, 2001; Arenz et al., “Synthese des ersten selektivenirreverilben Inhibitors der neutralen Sphingomyelinase”, Angew Chem.,112:1498-1500, 2000; Tanaka et al., “Structural Elucidation ofScyphostatin, an Inhibitor of Membrane-Bound Neutral Sphingomyelinase”,J. Am. Chem. Soc. 199:7871-7872, 1997; Saito et al., “AbsoluteConfiguration of Scyphostatin”, Organic Letters, 2:505-506, 2000; Hoyeet al., “Synthesis (and Alternative Proof of Configuration) of theScyphostatin C(1′)-C(20′) Trienoyl Fragment”, Organic Letters,2:1481-1483, 2000; Izuhara et al., “Studies toward the Total Synthesisof Scyphostatin: First Entry to the Highly Functionalized CyclohexenoneSegment”, Organic Letters, 3:1653-1656, 2001; Runcie et al., “A Shortand Efficient Route to Novel Scyphostatin Analogues”, Organic Letters,3:3237-3239, 2001; Chau et al., “Synthesis of Simple Aryl NeutralSphingomyelinase Inhibitors”, Asbtr. Pap.—Am. Chem. Soc., 2001; Arenz etal., “Manumycin A and Its Analogues Are Irreversible Inhibitors ofNeutral Sphingomyelinase”, ChemiBiochem., 2:141-143, 2001; Zeeck et al.,“Manumycin derivatives and the use thereof”, U.S. Pat. No. 5,079,263,issued Jan. 7, 1992; and Patel et al., “Manumycin Compounds”, U.S. Pat.No. 5,444,087, issued Aug. 22, 1995.

[0359] Other inhibitors of SMase that may serve as therapeutic agents orlead compounds, or may provide a chemical framework for preparingfocused chemical libraries that can be screened for inhibitors of SMase,include but are not limited to L-carnitine (Andrien-Abadie et al., FASEBJ. 13:1501-1510, 1999), and related compounds (U.S. Pat. No. 6,284,798);ubiquinol and ubiquinone homologs (Martin et al., J. Bioenerg Biomember33:143-153, 2001); antioxidants such as ascorbate and alpha-tocoperol(Hernandez et al., Circ. Res. 86:198-204, 2000); glutathione (oxidizedform) (Liu et al., J. Biol. Chem. 272:16281-16287, 1997; Liu et al., J.Biol. Chem. 273:11313-11320, 1998; Yoshimura et al., J. Neurochein.73:675-683, 1999; and Yoshimura et al., “Inhibition of NeutralSphingomyelinase Activation and Ceramide Formation by Glutathione inHypoxic PC12 Cell Death”, Journal of Neurochemistry, 73:675-683, 1999);Alutenusin, a protein produced by Penicillium spp. (Uchida et al., J.Antibiotics 52:572-574, 1999); SR 3357(2-isopropyl-1-4-[3-N-methyl-N-3,4-dimethoxy-phenethylamino] propyloxybenzenesulfonylindolizine) (Higuchi et al., J. Immunol. 157:297-304,1996; Lee et al., Biochem J. 334:457-461, 1998); desipramine (Lee etal., Biochem J. 334:457-461, 1998; Xu et al., J. Biol. Chem.273:16521-16526, 1998); DTT (Yamanaka et al., J. Neurochem.38:1753-1764, 1982); sphingomyelin methylene analogs (Hakogi et al.,Stereocontrolled synthesis of a sphingomyelin methylene analogue as asphingomyelinase inhibitor, Org Lett 2:2627-2629, 2000); and substitutedamino acids (U.S. Pat. No. 6,306,911 B1 to Wachter et al. Published PCTapplications WO 99/41265 and WO 00/58491 disclose compounds namedKF-1040A, KF-1040B, KF-1040T4A, KF-1040T4B, KF-1040T5A and KF-10407TB,which are stated to be inhibitors of SMase. Other SMase inhibitors aredisclosed in published PCT application WO 00/72833 A2. Sphingomyelinderivatives that are inhibitors of SMase are disclosed by Lister et al.(Biochimicha et Biophysica Acta 1256:25-30, 1995); Tazabekova et al.(Bioorg Khim 1987 May;13:648-653, 1987); and Hakogi et al.(Stereocontrolled synthesis of a sphingomyelin methylene analogue as asphingomyelinase inhibitor, Org Lett 2:2627-2629, 2000).

[0360] Pharmaceutical compositions of these and other inhibitors ofSMase, especially those that are formulated for rapid cardiac delivery,are used for this form sphingolipid-based cardiovascular therapy.

[0361] Desaturase catalyzes the conversion of dihydroceramide toceramide (Rxn. #9 in FIG. 2). Dihydroceramidase desaturase are disclosedin Geeraert et al., “Conversion of dihydroceramide into ceramide:involvement of a desaturase”, Biochem J., 327:125-132, 1997; Triola etal., “Synthesis of a Cyclopropene Analogue of Ceramide, a PotentInhibitor of Dihydroceramide Desaturase”, Angew. Chem. Int. Ed.,40:1960-1962, 2001; Heinz et al., “Sphingolipid-Desaturase”,PCT/DE99/01859, published as WO00/00593 on Jan. 6, 2000; Michel et al.,“Characterization of Ceramide Synthesis: A Dihydroceramide DesaturaseIntroduces The 4,5-TRANS-Double Bond of Sphingosine at the Level ofDihydroceramide”, 272:22432-22437, 1997. Inhibitors of desaturase aredisclosed in Triola et al., “Synthesis of a Cyclopropene Analogue ofCeramide, a Potent Inhibitor of Dihydroceramide Desaturase”, Angew.Chem. Int. Ed., 40:1960-1962, 2001.

[0362] Pharmaceutical compositions of inhibitors of desaturase,especially those that are formulated for rapid cardiac delivery, areused for this form sphingolipid-based cardiovascular therapy.

[0363] Cerebrosidases catalyze the production of ceramide fromglucosylceramide (Rxn. #14 in FIG. 2). Pharmaceutical compositions ofinhibitors of one or more cerebrosidases, especially those that areformulated for rapid cardiac delivery, are used for this form ofsphingolipid-based cardiovascular therapy.

[0364] Stimulation of Destruction of CER

[0365] The stimulation of enzymes that catalyze reactions that degradeCER (i.e., reactions that have CER as a reactant) is expected to resultin the stimulation of degradation of CER molecules. In general, it ispreferable that such reactions do not yield a undesirable, toxic and/orcardiotoxic sphingolipid, such as SPH, as a product (an example of anenzyme of this type is ceramidase). Other enzymes that may be stimulatedto enhance the degradation of CER include but are not limited to thefollowing:

[0366] Sphingomyelin Synthase catalyzes the conversion of ceramide tosphingomyelin (Rxn. #6 in FIG. 2); see, e.g., Luberto, et al.,“Sphingomyelin synthase”, a potential regulator of intracellular levelsof ceramide and diacylglycerol during SV40 transformation. Doessphingomyelin synthase account for the putativephosphatidylcholine-specific phopholipase C? PubMed, J. Biol Chem,273:14550-14559, 1998. Inhibitors of SM synthase include sphingomyelinmetabolites; Vivekananda et al., “Sphingomyelin metabolites inhibitsphingomyelin synthase and CTP:phophocholine cytidylyltransferase”, AmJ. Physiol Lung Cell Mol Physiol, 2281 :L91-L107, 2001. CER kinases aredisclosed in Bajjalieh et al., “Ceramide Kinase”, Methods in Enzymology,311:207-215, 1999; Kolesnick et al., “Characterization of a CeramideKinase Activity from Human Leukemia (HL-60) Cells: Separation FromDiacylglycerol Kinase Activity”, The Journal of Biological Chemistry,265:18803-18808, 1990. Pharmaceutical compositions of stimulators ofsphinomyelin synthase, especially those that are formulated for rapidcardiac delivery, are used for this form of sphingolipid-basedcardiovascular therapy.

[0367] Ceramide (CER kinase) catalyzes the conversion of ceramide toceramide-1-P (Rxn. #16 in FIG. 2). Pharmaceutical compositions ofstimulators of CER kinase, especially those that are formulated forrapid cardiac delivery, are used for this form of sphingolipid-basedcardiovascular therapy.

[0368] Glucosylceramide Synthase catalyzes the conversion of ceramide toglucosylceramide (Rxn. #13 in FIG. 2). For reviews, see Shayman et al.,“Glucosylceramide Synthase: Assay and Properties”, Methods inEnzymology, 311:42-49, 1999 and Marks et al., “Methods for StudyingGlucosylceramide Synthase”, Methods in Enzymology, 311:50-59, 1999.Pharmaceutical compositions of stimulators of glucosylceramide synthase,especially those that are formulated for rapid cardiac delivery, areused for this form of sphingolipid-based cardiovascular therapy.

[0369] Inhibitors of glucosylceramide synthase areknown. See U.S. Pat.No. 6,051,598 to Shayman et al.; Rani et al., “Cell Cyle Arrest Inducedby an Inhibitor of Glucosylceramide Synthase”, The Journal of BiologicalChemistry, 270:2859-2867, 1995; Abe et al., “Use of Sulfobutyl Etherβ-Cyclodextrin as a Vehicle forD-threo-1-Phenyl-2-decanoylamino-3-morpholinopropanol-RelatedGlucosylceramide Synthase Inhibitors”, Analytical Biochemistry,287:344-347, 2000; Oshefski et al., “Glucosylceramide SynthaseInhibition Enhances Vincristine-Induced Cytotoxicity”, Int. J. Cancer,93:131-138, 2001; Abe et al., “Glycosphingolipid depletion in Fabrydisease lymphoblasts with potent inhibitors of glucosylceramidesynthase”, Kidney International, 57:446-454, 2000; Lee et al., “ImprovedInhibitors of Glucosylceramide Synthase”, The Journal of BiologicalChemistry, 274:14662-14669, 1999; Abe et al., “Structural andstereochemical studies of potent inhibitors and glucosylceramidesynthase and tumor cell growth”, Journal of Lipid Research, 36:611-621,1995; Shayman et al., “Inhibitors of Glucosylceramide Synthase”, Methodsin Enzymology, 311:373-387, 1999; Jimbo et al., “Development of a NewInhibitor of Glucosylceramide Synthase”, J. Biochem 127:485-491, 2000;and Marks et al., “Methods for Studying Glucosylceramide Synthase”,Methods in Enzymology, 311:50-59, 1999.

[0370] Enzymes that catalyze the production of galactosylceramide fromCER (Rxn. #15 in FIG. 2) are stimulated to enhance the degradation ofCER. Pharmaceutical compositions of stimulators of such enzymes,especially those that are formulated for rapid cardiac delivery, areused for this form of sphingolipid-based cardiovascular therapy.

Example 10

[0371] Modulation of Metabolic Precursor of Cer

[0372] The concentration of harmful sphingolipids is lowered byinhibiting reactions that yield metabolic precursors of ceramide (CER),which is a metabolic precursor of SPH and S-1-P. Enzymes that catalyzesuch reactions include but are not limited to the following.

[0373] Inhibition of Production of Metabolic Precursors of CER

[0374] The concentration of harmful sphingolipids is lowered byinhibiting reactions that yield metabolic precursors of ceramide (CER),which is a metabolic precursor of SPH and S-1-P. Enzymes that catalyzesuch reactions include but are not limited to the following.

[0375] Serine Palmitoyl Transferase catalyzes the production of3-ketosphinganine (Rxn. #12 in FIG. 2), a precursor in ceramidesynthesis. For a review, see Dickson et al., “SerinePalmitoyltransferase”, Methods in Enzymology, 311:1-9, 1999.

[0376] Inhibitors of serine palmitoyl transferase include but are notlimited to viridiofungins (Mandala et al., J. Antibiot. (Tokyo)50:339-343, 1997; and Mandala et al., “Isolation and Characterization ofNovel Inhibitors of Sphingolipid Synthesis: Australifungin,Viridiofungins, Rustmicin, and Khafrefungin, Methods in Enzymology,311:335-348, 1999), lipoxamycin (Mandala et al., J. Antibiot. (Tokyo)47:376-379, 1994), and sphingofungins E and F (Horn et al., J. Antibiot.(Tokyo) 45:1692-1696, 1992). Other SPT inhibitors are disclosed byHanada et al., “Specificity of Inhibitors of Serine Palmitoyltransferase(SPT), a Key Enzume in Sphingolipid Biosynthesis, in Intact Cells”,Biochemical Pharmacology, 59:1211-1216, 2000; Zweerink et al.,“Characterization of a Novel, Potent, and Specific Inhibitor of SerinePalmitoyltransferase”, The Journal of Biological Chemistry,267:25032-25038, 1992; and Riley et al., “Fermentation, PartialPurification, and Use of Serine Palmitoyltransferase Inhibitors fromIsaria (=Cordyceps) sinclairii, Methods in Enzymology, 311:348-361,1999.

[0377] Pharmaceutical compositions of inhibitors of serine palmitoyltransferase, especially those that are formulated for rapid cardiacdelivery, are used for this form of sphingolipid-based cardiovasculartherapy.

[0378] 3-Ketosphiganine Reductase catalyzes the production ofsphinganine (dihydrosphingosine) (Rxn. #11 in FIG. 2), a precursor inceramide synthesis. See Beeler et al., “The Saccharomyces cerevisiaeTSC10/YBR265ωGene Encoding 3-Ketosphinganine Reductase Is Identified ina Screen for Temperature-sensitive Suppressors of the CA²⁺-sensitivecsg2Δ Mutant”, The Journal of Biological Chemistry, 273:30688-30694,1998. Pharmaceutical compositions of inhibitors of 3-ketosphiganinereductase, especially those that are formulated for rapid cardiacdelivery, are used for this form of sphingolipid-based cardiovasculartherapy.

[0379] Dihydroceramide Synthase catalyzes the acetylation ofdihydrosphingosine (Rxn. #10 in FIG. 2) which leads to the production ofdihydroceramide, a direct precursor of ceramide. Without wishing to bebound by any particular theory, dihydroceramide synthase may be the sameenzyme as ceramide synthase (Rxn. #4 in FIG. 2).

[0380] Inhibitors of ceramide synthase include Fumonisin B1 (a fungaltoxin) (Merrill et al., J. Lipid Res. 26:215-234A, 1993; Wang et al.,Adv. Lipid Res. 26:215-234, 1993; Tsunoda et al., J. Biochem. Mol.Toxicol. 12:281-289, 1998); derivatives of fumonisin (Humpf et al., J.Biol. Chem. 273:19060-19064, 1998); alternaria toxins (Id. and Mandalaet al., J. Antibiot. 48:349-356, 1995); viridiofungins (Merrill et al.,J. Lipid Res. 26:215-234A, 1993); astralifungins (Mandala et al., J.Antibiot. 48:349-356, 1995; Furneisen et al., Biochim. Biophys. Acta.1484:71-82, 2000); and D-erythro-N-myristoyl 2-amino-1-phenylpropanol(Hunnan, Science 274:1855-1859, 1996). Pharmaceutical compositions ofinhibitors of dihydroceramide synthase, especially those that areformulated for rapid cardiac delivery, are used for this form ofsphingolipid-based cardiovascular therapy.

[0381] Stimulation of Destruction of Metabolic Precursors of CER

[0382] The concentration of harmful sphingolipids is lowered bystimulating reactions that degrade metabolic precursors of ceramide(CER), which is a metabolic precursor of SPH and S-1-P. Enzymes thatcatalyze such reactions include but are not limited to the following.

[0383] Sphingomyelin Deacylase (SM deacylase) catalyzes the productionof sphingoylphosphorylcholine (SPC) from sphingomyelin (Rxn. #8 in FIG.2; see also FIG. 1). Pharmaceutical compositions of stimulators of SMdeacylase, especially those that are formulated for rapid cardiacdelivery, are used for this form of sphingolipid-based cardiovasculartherapy.

Example 11

[0384] An Inhibitor of Sphingomyelinase Blocks Hypoxia-InducedProduction of Sphingosine in a Cellular Model

[0385] L-camitine is a known inhibitor of SMase (Andrien-Abadie et al.,FASEB J. 13:1501-1510, 1999; see also U.S. Pat. No. 6,284,798). ThisExample demonstrates that L-camitine blocks the hypoxia-inducedproduction of sphingosine in a cellular model.

[0386] Cardiomyocytes were cultured on plastic dishes that were placedin a humidified modular incubator chamber (ICN Biomedicals, Aurora,Ohio.) pressurized to 0.04-0.05 psi by the particular gas mixture usedin the treatment. Warm (37° C.) Tyrode's solution containing 0.2 mM BSA,ampicillin (50 mg/mL), kanamycin (100 mg/mL) and fungizone (20 Mg/mL)was gassed for 15 minutes with 95%N2/5%CO2 prior to cell treatment. ThepO2 was monitored by a Micro pO2 System oxygen electrode (Lasar ResearchLabs, Los Angeles, Calif.) and found to be 4.0 mmHg for the duration ofthe hypoxia condition. The chamber was maintained at 37° C. for thedesignated times. Control (normoxia) cells were treated the same exceptfor the use of 95%O2/5%CO2 and incubated in a standard incubator. ThepO2 of the normoxia treatment was 7.3 mmHg. The pH of control andexperimental cell cultures was monitored with a micro pH electrode(Beetrode pH Electrodes, Sarasota, Fla.) and remained constant atpH=7.26+/−0.02 for normoxic cells and 7.15+/−0.03 for hypoxic cellsthroughout the experiment. Adult cardiomyocytes were cultured undereither normoxia or hypoxia conditions for 5 hours.

[0387] The cell-conditioned Tyrode's solution was collected and thecells were scraped from the culture dish. Both cellular andextracellular sphingolipids were extracted and then quantified byreverse-phase HPLC. The retention times of the key sphingolipids (SPH,SPC, S1P, DHSPH) followed in these experiments are shown in FIG. 4.Hypoxia resulted in a substantial increase in a peak corresponding toD-erythrosphingosine (SPH).

[0388] The fold-increases in SPH and TNF-alpha (inset) in response tohypoxia are shown. Cells were pretreated for 30 min with eitherTNFRII:Fc (0.5 ng/mL) or L-carnitine (20 ng/mL) prior to hypoxia. Dataare means+/−SEM from 19 separate experiments. FIG. 4 shows the amount ofSPH and TNF-alpha (inset) released into the cell-conditioned media andis expressed in terms of fold increases associated with hypoxia. Thesedata demonstrate that the SPH response (˜20-fold increase over normoxia)was more profound than the TNF-alpha response (˜10-fold increase overnormoxia, inset). Further, FIG. 4 shows that both TNFRII:Fc and thesphingomyelinase inhibitor L-camitine were capable of significantly(p<0.01) reducing the amount of total SPH.

Example 12

[0389] An Inhibitor of Sphingomyelinase Blocks the Effects of GlobalIschemia in an Animal Model

[0390] A rat model of ischemia and reperfusion was used to evaluate thetherapeutic potential of a gentamicin, an amino glycoside that inhibitssphingomyelinase.

[0391] Reagents

[0392] Krebs-Henseleit (KH) Buffer was prepared by combining thefollowing components: Chemical Conc. (mM) Amount per liter (g) NaHCO325.0 2.10  NaCl 118 6.90  KCl 4.7 0.35  MgSO4 (anhy) 1.2 0.145 NaH2PO41.2 0.145 CaCl2 1.2 0.175 Glucose 11 1.98 

[0393] The following steps are taken to prepare KH buffer. First, 95%O2/5% CO2 is bubbled through sterile water (Gibco Cat. Nos. 15230-147),which is concurrently warmed to 37° C. Second, the above ingredients areadded and the pH is adjusted to 7.4. Finally, the buffer is sterilizedby filtration.

[0394] Gentamicin vehicle solution was prepared as follows. A stocksolution of 0.1 M Gentamicin was prepared by adding 70 mg of GentamicinSulfate (Sigma, G1264) to 1 ml of KH Buffer. KH buffer (150 ml) wascombined with 150 μl)l of the stock solution to yield a 0.1 mM solution.

[0395] Protocol

[0396] The protocol used was essentially that described by Sakai et al.(A device for recording left ventricular contraction andelectrocardiogram in nonworking isolated perfused rat heart, Jpn JPharmacol 28:223-9, 1978), and Zelinski et al., (Phosphatidylcholinebiosynthesis in isolated hamster heart, J Biol Chem. 255:11423-8, 1980)with the exception, in the latter instance, that rat studies were usedin the experiments described herein instead of hamster hearts.

[0397] In brief, rats were anesthetized and their hearts were excised.The isolated hearts were cannulated and attached to Langendorffperfusion apparatus, and perfused with KH buffer. The hearts wereallowed to stabilize for 5-10 min. After stabilization, the heartsshould be paceable and drip rate should be between 5 and 10 mls perminute.

[0398] A small hole was cut in the left atrium of each animal to exposethe mitral valve. A deflated transducer balloon was introduced into theleft ventricle through the hole in left atrium. The transducer tube wastaped and adjusted so that there was no pressure on the heart. Leftventricular pressure was measured and recorded using the MacLab program.The transducer balloon was filled by adjusting amount of water using asyringe attached to the transducer via a stop-cock so that diastolicpressure was between 0 and 5 mm Hg. The hearts were allowed to stabilizefor 10 min. Once stabilized, the hearts should be paceable with aminimum of 100 beats per minute (bpm). The systolic pressure should beat least about 80 mm Hg, resulting in a developed left ventricularpressure of at least about 80 mm Hg.

[0399] The flow of KH buffer was switched to Gentamicin vehicle solutionusing the stop-cock at the top of the canula. The Gentamicin vehiclesolution was allowed to recirculate for 30 min., after which time two1.5 mL aliquots were collected and flash frozen in liquid nitrogen.

[0400] Next, all perfusate was turned off for 40 min. in order to beginglobal ischemia. The set up of the Langendorf apparatus was adjusted sothat the peristaltic pump was securely attached and all the air wasmoved out of the line to begin reperfusion. The flow of KH buffer wasturned off after 40 min., and two 1.5 mL aliquots of perfusate drippingfrom the heart were collected and flash frozen. The heart was thenreperfused for 45 min. Perfusate was collected at 2 min. intervals andflash frozen.

[0401] After about 45 min., the perfusate was turned off and theventricular pressure was no longer recorded. The atria were cut off, thepressure transducer was removed, and the ventricles were frozen in a 50mL conical tube.

[0402] The results, shown in FIG. 5, show that the neutralsphingomyelinase inhibitor gentamicin mitigated ischemia and reperfusioninjury in the Langendorff rat heart model of ischemia.

Example 13

[0403] HTS Screeening for Therapeutic Agents that InhibitSphingomyelinase

[0404] This Example describes an exemplary screening assay of theinvention that is used to screen for and identify compounds useful inthe therapeutic methods of the invention. The screening assay of thisExample, which is designed to isolate inhibitors of sphingomyelinase(SMase) from a library of compounds based on the structure ofaminoglycosides, may be in used high throughput screening (HTS) formats.

[0405] This Example describes a high-throughput screening (HTS)scintillation proximity assay (SPA) designed to identify aminoglyosidicinhibitors of sphingomyelinase (SMase). The HTS-SPA of the Exampleinvolves high-volume, homogeneous radiometric assays, based upon theprinciple of scintillation proximity. Although assays of a single plateare described in this Example, it is understood that many such plateswould be prepared and tested in high-throughput screening.

[0406] Sphingomyelinases

[0407] Sphingomyelinase (sphingomyelin choline phosphohydrolase) (SMase)catalyzes the hydrolytic cleavage of sphimgomyelin (SM) via reactionwhich lead to ceramide and phosphocholine generation (Brady, R. O.,Kanfer, J. N., Moek, M. B., and Fredrickson, D. S. 1966. The metabolismof sphingomyelin. II. Evidence of an enzymatic deficiency inNiemann-Pick disease. Proc. Natl. Acad. Sci. USA 55:366). Anysphingomyelinase can be used in the HTS assays, although mammalianSMases, particularly neutral SMases, are of particular interest.

[0408] Several different types of mammalian SMases have been identified,i.e.,

[0409] (1) Neutral, membrane-associated, Mg2+-stimulated SMases foundpredominantly in brain and kidney (Spence, M. W. 1993.Sphingomyelinases. Adv. Lipid Res. 26:3-23), which are known to arisefrom a separate gene from lysosomal SMase (Gatt, S., Dinur, T., andKopolovic, J. 1978. Niemann Pick disease: presence of themagnesium-dependent sphingomyelinase in brain of the infantile form ofthe disease. J. Neurochem. 31:547-550). For review of neutral SMases,see Chatterjee, “Neutral Sphingomyelinase”, Advances in Lipid Research,26:25-49, 1993; and Chatterjee, “Neutral sphingomyelinase: past, presentand future”, Chemistry and Physics of Lipds, 102: 79-96, 1999. At leasttwo structurally different types of mammalian neutral SMase (nSMase)have been cloned:

[0410] (a) The nSMase described by Stoffel et al., i.e., a cloned humanneutral SMase described in Tomiuk et al., Cloned mammalian neutralsphingomyelinase: Functions in sphingolipid signaling? Proc. Natl. Acad.Sci. (U.S.A.) 95: 3638-3643; Hofman et al., Cloning and characterizationof the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinaseProc. Natl. Acad. Sci. (U.S.A.) 97:5895-5900, 2000; and published PCTapplication WO 99/07855 to Stofel et al., entitled “NeutralSphingomyelinase,” was filed Aug. 11, 1998 and was published on Feb. 18,1999.

[0411] (b) The nSMase described in U.S. Pat. No. 5,919,687 toChatterjee, entitled “Recombinant N-SMases and Nucleic Acids EncodingSame”, Published PCT application WO 98/28445 to Chatterjee, entitled“Recombinant N-SMases and Nucleic Acids Encoding Same”, and Chatterjee,Molecular Cloning, Characterization, and Expression of a Novel HumanNeutral Sphingomyelinase, J. Biol. Chem. 274:37407-37412, 1999). In thelatter reference, the nSMase of Chatterjee is stated to be unrelated tothe nSMase of Stoffel et al. as the two nSMases have different aminoacid sequences (p. 37412, left column, 11. 33-34).

[0412] (2) Lysosomal SMases that act optimally at low pH and show nodependence on divalent cations (Kanfer, J. N., Young, O., Shapiro, D.,and Brady, R. O. 1966. The metabolism of sphingomyelin. I. Purificationand properties of a sphingomyelin-cleaving enzyme from rat liver tissue.J. Biol. Chem. 241:1081; Levade, T., Salvayre, R., and Blazy-Douste, L.1986. Sphingomyelinases and Niemann-Pick disease. J. Clin. Chem.Biochem. 24:205-220);

[0413] (3) Acidic, Zn2+-stimulated SMases present in fetal bovine serumand to a lesser degree in newborn human serum (Spence, M. W., Byers, D.M., Palmer, F. B. St. C., and Cook, H. W. 1989. A new Zn2+-stimulatedsphingomyelinase in fetal bovine serum. J. Biol. Chem. 264:5358-5363),and also secreted by several human cell types during severalpathophysiological processes (Schissel, S. L., Shuchman, E. H.,Williams, K. J., and Tabas, 1. 1996. Zn2+-stimulated sphingomyelinase issecreted by many cell types and is a product of the acidsphingomyelinase gene. J. Biol. Chem. 271:18431-18436);

[0414] (4) Cytosolic SMases that, like Mg2+-dependent neutral SMases,have a neutral pH optimum but no dependence on divalent cations(Okazaki, T., Bielawska, A., Domae, N., Bell, R. M., and Hannun, Y. A.1994. Characteristics and partial purification of a novel cytosolicmagnesium-independent, neutral sphingomyelinase activated in the earlysignal transduction of 1 a,25-dihydroxyvitamin D3-induced HL-60 celldifferentiation. J. Biol. Chem. 269:4070-4077).

[0415] (5) Placental SMases (Garcia-Ruiz, Human placentasphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase,induces oxidative stress, glutathione depletion, and apoptosis in rathepatocytes, Hepatology 32:56-65, 2000).

[0416] (6) Brain-specific SMases have been described (Yamanaka et al., JNeurochem. 38:1753-1764, 1982); Hofmann et al., “Cloning andcharacterization of the mammalian brain-specific, Mg²⁺-dependent neutralsphingomyelinase”, PNAS, 97:5895-5900, 2000; Bernardo et al.,“Purification and Characterization of a Magnesium-dependent NeutralSphingomyelinase from Bovine Brain”, The Journal of BiologicalChemistry, 275:7641-7647, 2000; Liu et al., “Purification andCharacterization of a Membrane Bound Neutral pH OptimumMagnesium-dependent and Phosphatidylserine-stimulated Sphingomyelinasefrom Rat Brain”, The Journal of Biological Chemistry, 273:34472-34479,1998. A rat brain SMase is commercially available (MDS Panlabs).

[0417] SMases from non-mammalian species include:

[0418] (1) Bacterial SMases, such as the well-characterizedPhospholipase C from Bacillus cereus (Ikezawa et al., Studies onsphingomyelinase of Bacillus cereus. I. Purification and properties,Biochim Biophys Acta February 1978 27;528(2):247-56; Hetland et al.,Phospholipase C from Bacillus cereus has sphingomyelinase activity,Scand J Clin Lab Invest February 1982;42(1):57-61; Gilmore et al., ABacillus cereus cytolytic determinant, cereolysin AB, which comprisesthe phospholipase C and sphingomyelinase genes: nucleotide sequence andgenetic linkage, J Bacteriol February 1989;171(2):744-53; Yamada et al.,Nucleotide sequence and expression in Escherichia coli of the genecoding for sphingomyelinase of Bacillus cereus , Eur J Biochem August1988 1;175(2):213-20; Johansen et al., Bacillus cereus strain SE-1:nucleotide sequence of the sphingomyelinase C gene, Nucleic Acids Res.16:103770, 1998; Fujii et al., Mg2+binding and catalytic function ofsphingomyelinase from Bacillus cereus , J Biochem (Tokyo) 124:1178-1187,1998; Gavrilenko et al., Nucleotide sequence of phospholipase C andsphingomyelinase genes from Bacillus cereus BKM-B164, Bioorg Khim19:133-138, 1993; Tomita et al., Secondary structure of sphingomyelinasefrom Bacillus cereus , J Biochem (Tokyo) 108:811-815, 1990; and Tamuraet al., Mass production of sphingomyelinase of Bacillus cereus by aprotein-hyperproducing strain, Bacillus brevis 47, and its purification,J Biochem (Tokyo) 112:488-491, 1992).

[0419] Other bacterial SMases are known and include, by way ofnon-limiting examples, those from Helicobacter pylori (Chan et al.,Purification and characterization of neutral sphingomyelinase fromHelicobacter pylori , Biochemistry 39:4838-4845, 2000; Lin et al.,Identification of neutral and acidic sphingomyelinases in Helicobacterpylori , FEBS Lett 423:249-253, 1998); Listeria ivanovii (Gonzalez-Zomet al., The smcL gene of Listeria ivanovii encodes a sphingomyelinase Cthat mediates bacterial escape from the phagocytic vacuole, MolMicrobiol 33:510-523, 1999); Staphylococcus aureus (Walev et al.,Selective killing of human monocytes and cytokine release provoked bysphingomyelinase (beta-toxin) of Staphylococcus aureus, Infect Immun64:2974-2979, 1996); and Clostridium perfringens (Saint-Joanis et al.,Gene cloning shows the alpha-toxin of Clostridium perfringens to containboth sphingomyelinase and lecithinase activities, Mol Gen Genet 1989Nov;219(3):453-60).

[0420] (2) Arachnoid SMases, such as the cytotoxic sphingomyelinase Dfrom the brown recluse spider (Loxosceles reclusa) (Gates et al., Serumamyloid p component: its role in platelet activation stimulated bysphingomyelinase d purified from the venom of the brown recluse spider(Loxosceles reclusa). Toxicon 28: 1303-1315, 1990).

[0421] (3) SMase homologs, such as ISC1 (YER019w) of Saccharomycescerevisiae (Sawai et al., Identification of ISC1 (YER019w) as InositolPhosphosphingolipid Phospholipase C in Saccharomyces cerevisiae, J BiolChem 275:39793-39798, 2000), which may have SMase activity in vitro eventhough sphingomyelin is not present in cells of the organism from whichthe SMase homolog is prepared. That is, an SMase homolog may act onsubstrates other than sphingomylein in vivo but nevertheless have bonafide SMase activity in vitro.

[0422] Other SMases that may be used to practice the invention includewithout limitation Romiti et al., “Characterization of sphingomyelinaseactivity released by thrombin-stimulated platelets”, Molecular andCellular Biochemistry, 205:75-81, 200; Sawai et al., “Function of theCloned Putative Neutral Sphingomyelinase as Lyso-platelet ActivatingFactor-Phospholipase C”, The Journal of Biological Chemistry, Vol. 274,Nos. 53, Dec. 31, 1999, pp. 38131-38139; Fensome et al., “A NeutralMagnesium-dependent Sphingomyelinase Isoform Associated withIntracellular Membranes and Reversibly Inhibited by Reactive OxygenSpecies”, The Journal of Biological Chemistry, 275:1128-1136, 2000;Holopainen et al., “Sphingomyelinase Activity Associated with HumanPlasma Low Density Lipoprotein”, The Journal of Biological Chemistry,275:16484-16489, 2000; and Hinkovska-Glacheva et al., “Activation of aPlasma Membrane-Associated Neutral Sphingomyelinase and ConcomitantCeramide Accumulation During IgC-Dependent Phagocytosis in HumanPolymorphonuclear Leukocytes”, Blood, 91:4761-4769, 1998.

[0423] Chemical Libraries Based on Aminoglycoside Structures

[0424] Combinatorial chemical libraries that are used in this screeningassay are preferably “biased” in the sense that they are prepared usingthe basic aminoglycoside structure as a framework to produceaminoglyosidic molecules having a multitude of positions where alternate“R” groups may be incorporated. The structure shown in FIG. 6, which has13 “R” groups, is used. In the following naturally occuringaminoglycoside antibiotics, the structure shown in FIG. 6 has thefollowing R groups that may be substituted; in all of these compounds,R11 is NH2, and R12 is OH, and R13 is OH. Aminoglycoside R1 R2 R3 R4 R5R6 R7 R8 R9 R10 Kanamycin A OH OH OH H NH2 H CH2OH OH H H Kanamycin BNH2 OH OH H NH2 H CH2OH OH H CH3 Kanamycin C NH2 OH OH H OH H CH2OH OH HH Gentamicin C1 NH2 H H NHCH3 NHCH3 H H CH3 OH CH3 Gentamicin C1a NH2 HH NH2 NH2 H H CH3 OH CH3 Gentamicin C2 NH2 H H NH2 NH2 H H CH3 OH CH3Gentamicin C26 NH2 H H NHCH3 NHCH3 H H CH3 OH CH3 Gentamicin B NH2 H HNH2 NH2 H H CH3 OH CH3 Tobramycin NH2 H OH H NH2 H H OH H H DibekacinNH2 H H H NH2 H H OH H H Aberkacin NH2 H H H NH2 COR' COR' OH H HIsepamicin OH OH OH H NH2 COR COR CH3 OH CH3

[0425] A combinatorial library of aminoglycosides and aminoglycosidederivatives is prepared using methods discussed in Sucheck et al.(Combinatorial synthesis of aminoglycoside libraries, Curr Opin DrugDiscov Devel 4:462-70, 2001); Hofstadler et al., (Multiplexed screeningof neutral mass-tagged RNA targets against ligand libraries withelectrospray ionization FTICR MS: a paradigm for high-throughputaffinity screening, Anal Chem 71:3436-40, 1999), and references citedtherein.

[0426] Pre-Assay Studies

[0427] A FlashPlate® microtiter plate (NEN Life Science Products,Boston, Mass.) is used in the assay. The interior of each well of themicrotiter plate is permanently coated with a thin layer ofpolystyrene-based scintillant that produces a signal when the surface ofthe well is in close proximity to any of a variety of isotopes (e.g.,3H, 125I, 14C and 33P). Because the scintillant is permanently bound tothe wells of the plate, a liquid scintillation cocktail does not need tobe added to the wells during the assay.

[0428] A radiolabeled substrate for SMase is coated or bound onto thesurface of wells in a FlashPlate. The radioactive decay associated withthe radiolabeled causes a microplate surface scintillation effectdetectable on a microplate scintillation counter. Radiolabel that isreleased from the radiolabeled substrate by the enzymatic activity ofSMase does not activate the scintillant. Thus, after SMase is added, thesignal from the radiolabel decreases over time. If an inhibitor of SMaseis present in a well, the radiolabel is released at a lower rate, andthe scintillant-mediated signal thus decreases at a slower rate.

[0429] In the present Example, the radiolabelled SMase substrate that iscoated onto the surface of wells in a FlashPlate is [3H]sphingomyelin(NEN Life Sciences). Before HTS is begun, studies are done to determinethe optimal means and conditions for coating the wells with thesubstrate. Aliquots of [3H]sphingomyelin, having concentrations rangingfrom 0.1 to 10 u Ci/ml, are prepared in a constant volume of Trisbuffer. The aliquots are added at 0.2 ml/well at room temperature andthe signal from the coated microtiter plates is read at regularintervals using a TopCount NXT Microplate Scintillation and LuminescenceCounter (Packard BioScience). Two parameters are optimized in thesestudies. First, the time at which the maximum counts/well is achievedbefore the signal hits a plateau; typically, this occurs after 10 hours.Second, the range of [3H]sphingomyelin that can be added to the wellsand still exhibit a linear increase in signal; typically, 0.2 uCi/wellis used.

[0430] Next, studies are done to determine the amount of time needed tohydrolyze the maximum amount of substrate at a chosen concentration. Aplate is coated using the optimized conditions described above, i.e.,0.2 uCi/well with 10 hours of pre-assay incubation. The SMase being usedin the assay is added (0.2 u/ml, diluted in PBS with 1 mg/ml CaC12 and 1mg/ml MgC12). The plate is incubated at room temperature and counts areread at regular time points over a period of 24 hours. The hydrolysis ofthe substrate occurs rapidly at first before reaching a plateau,typically after about the first 3 hours of incubation.

[0431] In these initial studies, and in plates used in HTS, severalcontrol wells are used. In one type of control, commerically available(Sigma) SMases are added to control wells to confirm the activity of theassay, including bacterial SMases (from Staphylococcus aureus orBacillus cereus ) or a human placental SMase. If the target SMase isdependent on a divalent cation, a chelator such as EDTA is added to oneor more control wells.

[0432] HTS Assays

[0433] A target SMase of interest is prepared by recombinant DNAtechnology. The target SMases in this Example are mammalian neutralSMases. The nSMase described by Chatterjee (U.S. Pat. No. 5,919,687) andthe nSMase described by Stoffel et al. (WO 98/28445) are both testedbecause they have unrelated amino acid sequences, and either or both maybe involved in sphingolipid metabolism and/or signaling incardiovascular tissues.

[0434] In the HTS-SPA, many microtiter plates are prepared as describedabove. In non-control wells, a member of the aminoglycoside chemicallibrary is added to each well, followed by the addition of the targetSMase, and the signals from the wells of the plates are read. When largenumbers of plates are to be screened, an external plate stacker can beadded to the TopCount NXT Microplate Scintillation and LuminescenceCounter, which allows for more than 15,000 samples to be loaded andtested unattended. Wells in which the signal from the unhydrolyzedsubstrate does not decrease, or decreases less rapidly than in a controlwell comprising the target SMase only, identify members of the librarythat are candidate inhibitors of the target SMase. The activity of thesecandidate compounds is confirmed by repeated testing.

[0435] The compounds are further characterized in terms of otherdesirable attributes. For example, the safety and efficacy of thecompositions and methods for sphingolipid-based cardiovascular therapyare initially evaluated in cell culture (e.g., cultured cardiomyocytes)and animal models. Non-limiting examples of such animal models includerat and pig models of infarction (Olivetti et al., Cardioscience6:101-106, 1995; Jacobs et al., J. Mol. Cell Cardiol. 31:1949-1959,1999; Gunther et al., Eur. J. Pharma. 406:123-126, 2000; and Holmes etal., Circulation 90:411-420, 1994).

Example 14

[0436] Modulation of the Sphingomyelin Signaling Pathway

[0437] The sphingomyelin signaling pathway (a.k.a. the SM pathway) is a“cascade” of biochemical events in which proteins in the pathway areactivated (by enzymatic chemical modification or otherwise) with the endresult that sphingosine metabolism is affected. In most instances,activation of the SM pathway leads to increased production of ceramide.For reviews of the molecular biology of the sphingomyelin signalingpathway, see Hannun et al., Adv. Lipid Res. 25:27-41, 1993; Liu et al.,Crit. Rev. Clin. Lab. Sci. 36:511-573, 1999; Igarashi, Y., J. Biochem.122:1080-1087, 1997; and Oral et al., J. Biol. Chem. 272:4836-4842,1997.

[0438] It has suggested that the sphingomyelin signal transductionpathway is activated during cardiac ischemia/hypoxia (Bielawska et al.,Am. J. Pathol. 151:1257-1263, 1997; Meldrum, Am. J. Pathol. 274:577-595,1998; and Cain et al., J. Mol. Cell. Cardiol. 31:931-947, 1999). If so,there must be a factor or process that mediates the ischemia-induced SPHproduction. The most likely candidate for the mediator is thepro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Invarious animal models of ischemia, the myocardium produces TNFα(Squadrito et al., Eur. J. Pharmacol. 237:223-230, 1993; Herrmann etal., European Journal of Clinical Investigation 28:59-66, 1998; Meldrum,Ann. Thorac. Surg. 65:439-443, 1998). Recent evidence demonstrates thatthe cardiomyocytes themselves produce TNFα and secrete the cytokine intothe extracellular fluid (Comstock et al., J. Mol. Cell Cardiol.30:2761-2775, 1998). Since TNFα receptors are expressed bycardiomyocytes (Krown et al., FEBS Letters 376:24-30, 1995; Torre-Amioneet al, Circulation 92:1487-1493, 1995), an autocrine/paracrine role forTNFα has been suggested (Meldrum, Ann. Thorac. Surg. 65:439-443, 1998).Significantly, TNFα induces SPH production and apoptosis in cardiacmyocytes (Krown et al., J. Clin. Invest. 98:2854-2865, 1996), presumablyby acting by binding to the cardiomyocyte complement of TNFα receptors.

[0439] Activation of the sphingomyelin signal transduction cascade is akey early event in the cytotoxic (apoptotic) effects of the cytokineTNFα (Zhang et al., Endo. 136:4157-4160, 1995). TNFα can causesignificant apoptosis in cultured rat cardiomyocytes and it has beensuggested that TNFα-induced SPH production is responsible for the celldeath triggered by TNFα (Krown et al, J. Clin. Invest. 98:2854-2865,1996).

[0440] Inhibitors of Cytokines that Activate the Sphingomyelin SignalingPathway

[0441] The SM pathway, many steps of which occur intracellularly, isinduced by a variety of extracellular stimuli. In sphingolipid-basedcardiovascular therapy, such stimuli are at least partially blocked. SMpathway-inducing agents that are desirably interfered with include butare not limited to cytokines. Cytokines of particular interest includebut are not limited to pro-inflammatory cytokines, interferons andchemokines. Pro-inflammatory cytokines of particular interest includebut are not limited to TNF-alpha; interleukins such as IL-1beta, IL-2,IL-10, interferons of particular interest include but are not limited togamma-IFN. Chemokines of particular interest are those involved in theischemic process including but not limited to interleukin-8 (IL-8), andthe monocyte chemotaxic proteins MCP-1 and MCP-2. One non-limitingexample of an agent that may be used to modulate the SM pathway (inparticular, stroke-induced apoptosis) is the immunosuppresant FK506(Herr et al., Brain Res. 826:210-219, 1999).

[0442] Agents that Block Cytokine-Induced Activation of the SM Pathway

[0443] Sphingolipid-based cardiac therapeutic agents that are used toinhibit the actions of cytokines include but are not limited to anantibody directed to a cytokine or to a cytokine receptor; a cytokinereceptor fragment that binds a cytokine but is otherwise biologicallyinactive; and a cytokine analog that binds cytokine receptor but isotherwise biologically inactive.

[0444] As one non-limiting example of this aspect of the invention, anantibody directed to a cytokine or a cytokine receptor is used as atherapeutic agent in sphingolipid-based cardiovascular therapy. Suchantibodies are generated in an immunoreactive response to a cytokine, acytokine receptor, or a synthetic polypeptide derived therefrom. Apreferred type of antibody is a monoclonal antibody, which is initiallyisolated from a hybridoma; more preferred is a monoclonal antibody thathas been “humanized” via molecular genetic manipulation. Also preferredare fragments, preferably soluble, which are derived from antibodies toa cytokine and retain the ability to bind a cytokine, such as, e.g,single-chain Fv analogs (scFv). The isolation, production, humanizationand derivatization of antibodies is described in Ramnarayan et al., Am.Biotechnol. Lab. :26-28, 1995; Gavilondo et al., BioTechniques29:128-145, 2000; Kling, J., Modern Drug Discovery 2:33-45, 1999;Morrow, K. J. Jr., American Laboratory 32:15-19, 2000; Huston et al,Methods in Enzymology 203:46-88, 1991; Johnson et al., Methods inEnzymology 203:88-98, 1991; Güssow et al., Methods in Enzymology203:99-121, 1991; and references cited therein.

[0445] A preferred antibody that is used as a therapeutic agent insphingolipid-based cardiovascular therapy is one that blocks the bindingof a cytokine to its receptor. Assays for determining the degree ofinhibition of binding of a cytokine to its receptor (see, e.g., Murataet al., Anal. Biochem. 282:11-120, 2000) are used in initial assessmentsof the effectiveness of such antibodies.

[0446] As another non-limiting example of this aspect of the invention,a receptor fragment that binds a cytokine but is otherwise biologicallyinactive is used as a therapeutic agent in sphingolipid-basedcardiovascular therapy. For example, therapeutic inhibition of the SMpathway is achieved by blocking the binding of extracellular TNF-alphato a cellular receptor (TNFR); this in turn prevents the activation ofthe SM pathway. The binding of TNF-alpha to a TNFR is directly orcompetitively inhibited. One example of an agent for competitiveinhibition of the binding is a soluble TNF-alpha receptor fragment suchas TNRFII:Fc. TNFRII:FC receptor fragment, which is sold as Embrel^(R)(from Immunex Corporation, Seattle, Wash.). This soluble fusion proteinwas made from the extracellular binding domain of the TNF type IIreceptor and an immunoglobulin Fc portion of IgG1. This soluble fusionprotein has a very high affinity for TNF (Im et al., J. Biol. Chem275:14281-14286, 2000).

[0447] As another non-limiting example of this aspect of the invention,small molecules that serve as inhibitors and/or antagonists ofsphingolipid receptors are used as therapeutic agents insphingolipid-based cardiovascular therapy. Such molecules includesumarin, which blocks EDG-3 action (Mandala et al., Proc. Natl. Acad.Sci. U.S.A. 97:7859-7864, 2000), and pertusis toxin, which blocks EDGreceptors that use Gi (Gonda et al., Biochem. J. 337:67-75, 1999).

[0448] In addition to TNF and Fas, other examples of extracellularagents that activate the SM pathway by interacting with their receptorsinclude but are not limited to Fas and the Fas receptor (Brenner et al.,J. Biol. Chem. 272:22173-22181, 1997; and nerve growth factor (NGF) andthe p75 neutrotrophin receptor (p75NTR) (Dobrowsky et al., Science265:1596-1599, 1994).

[0449] Other Agents Directed to the Sphingomyelin Signaling Pathway

[0450] Sphingolipid-based cardiovascular therapy is also achievedthrough the use of compounds that bind sphingolipid receptors thatinitiate and stimulate the sphingomyelin signaling pathway. This pathwayultimately results in increased ceramide production. An increased levelof ceramide would, in turn, be expected to result in elevatedconcentrations of undesirable sphingolipids such as, e.g., S-1-P andSPH. Such receptor-binding agents may be, by way on non-limitingexample, antibodies or antibody fragments, small (organic) molecules, orsphingolipid derivatives that bind the receptors but do active the SMAsignaling pathway. The synthesis of representative sphingolipidderivatives are described in, by way of non-limiting example, PCTpublished patent application WO 99/12890; U.S. Pat. Nos. 5,663,404 and6,051,598, and 5,260,288 and 5,391,800.

[0451] In other instances, as is explained in detailed elsewhere herein,components of the SM pathway are used to create therapeutic proteinsthat retain the ability to bind sphingolipids but are otherwisebiologically inactive. Moreover, various steps in the SM pathway arespecifically inhibited by dominant-negative derivatives of proteinsinvolved in a particular step in the cascade, antisense molecules andconstructs, and gene therapeutics.

Example 15

[0452] Sphingolipid-Binding Protein Derivatives

[0453] Sphingolipid-binding protein derivatives are used forsphingolipid-based cardiovascular therapy in one aspect of theinvention. Such protein derivatives retain the ability to bindsphingolipids, even if other functions, biochemical activities and/orcharacteristics of the protein are altered, compromised or absent in theprotein derivative. Protein derivatives may be oligopeptides synthesizedin vitro, proteins that have been purified from an animal and chemicallyor otherwise modified, proteins produced via recombinant DNA technology,or combinations thereof. Non-limiting examples of sphingolipid-bindingprotein derivatives include enzyme derivatives, and receptorderivatives. A sphingolipid-binding enzyme derivative can be, forexample, a noncatalytic derivative of an enzyme involved in sphingolipidmetabolism that retains the ability to bind sphingolipids. Asphingolipid binding receptor derivative can be, for example, a solublederivative of a membrane-bound sphingolipid receptor that bindssphingolipids, e.g., soluble derivatives of a member of the EDG orSCaMPER family of receptors. A “sphingolipid-binding protein derivative”may also be an antibody or antibody derivative; “antibody derivativeproteins” are antibody fragments that retain the ability to specificallybind sphingolipids. Such antibody fragments are, by way of non-limitingexample, single-chain FV analogs (scFv's), complementarity-determiningregions (CDR's), and the like, and fusion proteins comprising suchantibody fragments. See Gavilondo et al., BioTechniques 29:128-145,2000; and Verma et al., Journal of Immunological Methods 216:165-181,1998.

[0454] Such sphingolipid-binding protein derivatives can be, but neednot be, derived from an enzyme or receptor from the animal that isintended to be treated. Such derivatives may be prepared from homologousenzymes or receptors from a non-human mammal (e.g., a feline SMasederivative), or from analogous enzymes or receptors from an organismthat belongs to a different biological Family, Order, Class or Kingdom(e.g., an arachnid or bacterial SMase).

[0455] Sphingolipid-Binding Enzyme Derivatives

[0456] Enzymes from which biologically inactive (non-catalytic)sphingolipid-binding derivatives are obtained include but are notlimited to the following. Such derivatives of these enzymes bind theirsubstrate, which is a undesirable, toxic and/or cardiotoxicsphingolipid, and thereby lower the actual or available concentration ofthe sphingolipid, and/or render the sphingolipid biologically inactivewith respect to its cardiotoxic effects. Preferably, such derivativesare soluble and may be formulated into a pharmaceutical compositionsuitable for sphingolipid-based cardiovascular therapy.

[0457] S-1-P is bound by enzymes having S-1-P as a substrate, such as,by way of non-limiting example, S-1-P lyase and S-1-P phosphatase.Non-catalytic derivatives of these enzymes bind S-1-P and interfere withits harmful effects.

[0458] SPH is bound by enzymes having SPH as a substrate, such as, byway of non-limiting example, SPH Kinase and Ceramide synthase.Non-catalytic derivatives of such enzymes bind SPH and interfere withits harmful effects.

[0459] Ceramide is bound by enzymes having ceramide as a substrate, suchas, by way of non-limiting example, ceramidase, SM synthase, ceramidekinase, and glucosylceramide synthase. Non-catalytic derivatives of suchenzymes bind ceramide and interfere with the harmful effects of itsmetabolites, such as, in particular, SPH and S-1-P.

[0460] Sphingomyelin is bound by enzymes having sphingomyelin as asubstrate, such as, by way of non-limiting example, SMase. Non-catalyticderivatives of such enzymes bind sphingomyelin and interfere with theharmful effects of its metabolites such as, e.g., ceramide, SPH andS-1-P.

[0461] Sphingolipid-Binding Receptor Derivatives

[0462] Receptors from which biologically inactive (e.g., non-signaltransducing) sphingolipid-binding derivatives are obtained include butare not limited to the following. Such derivatives of these receptorsbind their ligand, which is a cardiotoxic sphingolipid, and therebylower the actual or available concentration of the sphingolipid, and/orrender the sphingolipid biologically inactive with respect to itscardiotoxic effects. Preferably, such derivatives are soluble and may beformulated into a pharmaceutical composition suitable forsphingolipid-based cardiovascular therapy.

[0463] EDG Receptors

[0464] Receptors that bind S-1-P are used in this aspect of theinvention (for a review of some S-1-P-binding receptors, see Spiegel etal., Biochim. Biophys. Acta 1484:107-116, 2000).

[0465] EDG-1 was the first identified member of a class of Gprotein-coupled endothelial-derived receptors (EDG). Such receptorsinclude but are not limited to members of the EDG family of receptors(a.k.a. 1pA receptors, Chun, Crit. Rev. Neuro. 13:151-168, 1999), andisoforms and homologs thereof such as NRG1 and AGR16.

[0466] For reviews, see Goetzl et al., Adv. Exp. Med. Biol. 469:259-264,1999; Chun et al., Cell. Biochem. Biophys. 30:213-242, 1999); Sato, “Anew role of lipid receptors in vascular and cardiac morphogenesis”, TheJournal of Clinical Investigation, 6:939-940, 2000.

[0467] EDG-1 is described by Lee et al, (Ann. NY Acad. Sci. 845:19-31,1998). Human EDG-1c genes and proteins are described in published PCTapplication WO 99/46277 to Bergsma et al. Described in Au-Young, et al.,U.S. Pat. No. No. 5,912,144, issued Jun. 15, 1999, “EDG-1 ReceptorHomolog”; Bergsma et al., WO 97/46277, published Sep. 16, 1999, “HumanEDG-1c Polynucleotides and Polypeptides and Methods of Use”; and Okamotoet al., “EDG1 Is a Functional Sphingosine-1-Phosphate Receptor That IsLinked via a G_(i/o) to Multiple Signaling Pathways, IncludingPhospolipase C Activation, Ca²+ Mobilization, Ras-Mitogen-activatedProtein Kinase Activation, and Adenylate Cyclase Inhibition”, TheJournal of Biological Chemistry, 273:27104-27110, 1998.

[0468] EDG-3 is described by Okamoto et al. (Biochem. Biophys. Res.Commun. 260:203-208, 1999) and An et al. (FEBS Letts. 417:279-282,1997). See also An et al., J. Biol. Chem. 275:288-296, 2000; EDG-3(a.k.a. LP(B4)); Tsui, U.S. Pat. No. 6,130,067, issued Oct. 10, 2000,“Human EDG3SB Gene”; and Siehler et al., “Sphingosine 1-PhosphateActivates Nuclear Factor-κB through Edg Receptors: Activation ThroughEdg-3 and Edg-5, but not Edg-1, in Human Embryonic Kidney 293 Cells,”JBC Papers in Press Published Oct. 22, 2001 as Manuscript M01107220.

[0469] EDG-5 human and mammalian genes are described in U.S. Pat. No.6,057,126 to Munroe et al. and published PCT application WO 99/33972 toMunroe et al. The rat homolog, H218 (a.k.a. ARG16) is described in U.S.Pat. No. 5,585,476 to MacLennan et al. Van Brocklyn et al, J. Biol.Chem. 274:4626-4632, 1999; and Gonda et al., Biochem. J. 337:67-75,1999. See also An et al., J. Biol. Chem. 275:288-296, 2000.

[0470] EDG-6 is described by Graler et al. (Genomics 53:164-169, 1998)and Yamazaki et al. (Biochem. Biophys. Res. Commun. 268:583-589, 2000).

[0471] EDG8 from rat brain is described by Im et al., (J. Biol. Chem.275:14281-14286, 2000). Homologs of EDG-8 from other species, includinghumans, may also be used.

[0472] The Mil Receptor (Mil is an abbreviation for “miles apart”) bindsS-1-P and regulates cell migration during vertebrate heart development.The Mil receptor of Zebrafish is described by Mohler et al. (J. Immunol.151:1548-1561, 1993). Another S-1-P receptor is NRG1 (nerve growthfactor regulated gene-1), the rat version of which has been identified(Glickman et al., Mol. Cel. Neurosci. 14:141 -152, 1999).

[0473] Receptors that bind SPC are also used in this aspect of theinvention. Such receptors include but are not limited to members of theSCaMPER family of receptors (Mao et al., Proc. Natl. Acad. Sci. U.S.A.93:1993-1996, 1996; Betto et al., Biochem. J. 322:327-333, 1997), andovarian cancer G-protein-coupled receptor 1; Xu et al.,“Sphingosylphopsphorylcoline is a ligand for ovarian cancerG-protein-coupled receptor 1”, Nature Cell Biology, 2:261-267, 2000.

[0474] Some evidence suggests that EDG-3 may bind SPC in addition toS-1-P (Okamoto et al., Biochem. Biophys. Res. Commun. 260:203-208,1999). Derivatives of EDG-3 that bind both S-1 -P and SPC are used inone aspect of the invention. Also usuable is the receptor described inAmes et al., WO 01/04139 A2, published Jan. 18, 2001, “Polynucleotideand Polypeptide Sequences of Human AXOR29 Receptor and Methods ofScreening for Agonists and Antagonists of the Interaction Between HumanAXOR29 Receptor and its Ligands”.

[0475] Receptors that bind LPA (lysophosphatic acid) are alos used inthis aspect of the invention. LPA receptors are described by Im et al.,“Molecular Cloning and Characterization of a Lysophosphatidic AcidRecetpor, Edg-7, Expressed in Prostate”, Molecular Pharmacology,57:753-759, 2000; An et al., “Characterization of a Novel Subtype ofHuman G Protein-coupled Receptor for Lysophosphotatidic Acid”, TheJournal of Biological Chemistry, 273:7906-7910, 1998; Fukushima et al.,“A single receptor encoded by vzg-1/lp_(A)/edg-2 couples to G proteinsand mediates multiple cellular responses to lysophosphatidic acid”,Proc. Natl. Acad. Sci., 95:6151-6156, 1998; Chun et al., “U.S. Pat. No.6,140,060 issued Oct. 31, 2000, “Closed Lysophosphatidic AcidReceptors”; and Kimura et al., “Two Novel Xenopus Homologs of MammalianLPEDG-2 Function as Lysophosphatidic Acid Receptor Xenopus Oocytes andMammalian Cells”, JBC Papers in Press Published on-line Feb. 5, 2001 asManuscript M011588200.

[0476] Soluble receptor fragments are derivatives of membrane-boundreceptors in which the transmembrane portions of the receptor have beenremoved. Receptor-derived polypeptides that are soluble, i.e., have losttheir transmembrane portion, and which retain their ability to bind thereceptor substrate, are solube receptor fragments that may havetherapeutic value as agents that bind undesirabel sphingolipids. Studiesthat have identified portions of Edg receptors that bind to S-1-P arehelpful as guides in designing soluble receptor fragments (Parrill etal., Identification of Edg1 Receptor Residues That Recognize Sphingosine1-Phosphate, The Journal of Biological Chemistry, 275:39379-39384, 2000;and Wang et al., A Single Amino Acid Determines LysophospholipidSpecificity of the S1P₁(EDG1) and LPA₁ (EDG2) Phospholipid Growth FactorReceptors, JBC Papers in Press Published on-line Oct. 16, 2001 asManuscript M107301200.

[0477] Sphingolipid-Binding Proteins from Natural Sources

[0478] Sphingolipid-binding proteins that may be used in the inventioninclude those isolated from natural sources. A non-limiting example ofsuch a protein is lysenin, a protein that binds sphingomyelin, which hasbeen prepared from the earthworm Eisenia foetida (Yamaji et al., J.Biol. Chem. 273:5300-5306, 1998).

Example 16

[0479] Cloning of Rat and Human Scamper Genes

[0480] The rat and human SCaMPER genes were obtained using a combinationof reverse transcription and polymerase chain reactions (RT-PCR) as isknown in the art (see, for example, PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich, ed., 1989).

[0481] Tissue was prepared from various human and rat sources asfollows. Human heart tissue from an expired heart failure patient wascollected, frozen in liquid nitrogen and stored at −70° C. Rat heart andskeletal tissue was freshly obtained from sacrificed adultSprague-Dawley rats. RNA from these tissues was isolated using thecommercially available Qiagen Rneasy Mini Kit according to themanufacturer's protocols (Qiagen, Valencia, Calif.).

[0482] PCR amplification of the target sequences was performed using thecommercially available Qiagen One-Step RT-PCR kit according to themanufacturer's protocols. Primers sets included:

[0483] 5′-CCAGGATTCATCATATGTTAAAAG-3′ (upper) (SEQ ID NO.: 1); and

[0484] 5′-ATCAGTGGGTGCATCAGTAGC-3′ (lower) (SEQ ID NO.: 2) for the openreading frame (ORF) and the 3′-sequence of SCaMPER (designed fromGenBank accession number U33628).

[0485] Amplification utilized cycling regimens according to themanufacturer's recommendations. Briefly, reactions were optimized using40 PCR cycles, each cycle consisting of 45 seconds denaturation at 95°C., 45 seconds of annealing at 50° C., and 1 minute of polymerization at72° C. The resulting PCR products were subcloned into pCR3.1 TOPO orpCDNA3.1-V5/HIS TOPO (Invitrogen, Carlsbad, Calif.) using standardtechniques. Sequencing with a T7 primer was performed at the San DiegoState University DNA Core Facility (San Diego, Calif.). The resultantSCaMPER sequences are presented as SEQ ID NO.: 3, the open reading frameof the rat SCaMPER gene and SEQ ID NO.: 4, the open reading frame of thethe human SCaMPER gene. Rat SCaMPER ORF (SEQ D NO.:3) 001 ATGTTAAAAGTGAGCAGGGT CTCAAGTGAA GGTTTAATAT CACTTTCTAT CACTGAGGCA 061 CCTGATCTTAAGATCAGGGA TCCTAAGATA GAGAAACTCT ACCTTCCAGT TTTTTATTTA 121 AATGCACACATCTACTTAAA TGCACTCAGT ACTCTCCTGA ACTCTCATTG TGGCGAGAAC 181 TGTTTTCATGGTTATGAAC AATTACAGAA TGCCACTTTT CCAGTTTGGA GAAATATATT 241 CATTTATATAAACAGGGTCA GGAACACCAA GAGGCAAGGA GGAGGGGGTG GTGTGAGTGG 301 GAAAGGTGAGATGAAGCAGT GCTTCCTCTC TTAA

[0486] Human SCaMPER ORF (SEQ ID NO.:4)ATGTTAAGTGAGCAGGGTCTCAAGTGAAGGTTTAATATCACTTTCTATCACTGAGGCACCTGATCTTAAGATCAGGGATCCTAAGATAGAGAAACTCTACCTTCCAGTTTTTTATTTAAATGCACACATCTACTTAAATGCACTCAGTACTCTCCTGAACTCTCATTGTGGCGAGAACTGTTTTCATGGTTATGAACAATTACAGAATGCCACTTTTCCAGTTTGGAGAAATATATTCATTTATATAAACAGGGTCAGGAACATCAAGAGGCAAGGAGGAGGGGGTGGTGTGAGTGGGAAGGTGAGATGAAGCAGTGCTTCCTCTCTTAA

Example 17

[0487] Cloning of Rat EDG-3 Genes

[0488] The rat Edg-3 gene was obtained using a combination of reversetranscription and polymerase chain reactions (RT-PCR) as is known in theart (see, for example, PCR Technology: Principles and Applications forDNA Amplification, H. A. Erlich, ed., 1989).

[0489] Rat heart, liver and skeletal muscle tissue was freshly obtainedfrom sacrificed rats. RNA from these tissues was isolated using thecommercially available Qiagen Rneasy Mini Kit according to themanufacturer's protocols (Qiagen, Valencia, Calif.).

[0490] PCR amplification of the target sequences was performed using thecommercially available Qiagen One-Step RT-PCR kit according to themanufacturer's protocols. Primers sets included:5′ -TTATGGCAACCACGCACGCGCAGG - 3′ (upper) (SEQ ID NO.:5) 5′ -AGACCGTCACTTGCAGAGGAC - 3′ (lower) (SEQ ID NO.:6)

[0491] Amplification utilized cycling regimens according to themanufacturer's recommendations. Briefly, reactions were started at 50°C. for 30 minutes, followed by 95° C. for 15 minutes; and then optimizedusing 40 PCR cycles, each cycle consisting of 45 seconds denaturation at95° C., 45 seconds of annealing at 63±5° C., and 1 minute ofpolymerization at 72° C. The resulting PCR products were subcloned intopCR3.1 vector using the Invitrogen TA cloning kit (Invitrogen, Carlsbad,Calif.) using standard techniques. Briefly, after RT-PCR, a 1% agarosegel was used to separate the PRC products from unused primers and dNTPselectrophoretically. The approximately 1200 bp fragment was then excisedquickly under UV light and the Bio101 Geneclean kit was then used topurify the DNA. The purified DNA was then ligated into the PCR3.1vector. The ligation mix was then transformed into Invitrogen Top10chemically competent cells with heat shock. Following a 1 hourincubation shaking at 37° C., the cells were spread on a LB platecontaining ampicillin and allowed to grow overnight at 37° C. Severalindividual colonies were chosen and used to inoculate culture tubescontaining 3 mls of LB-ampicillin media. After 8-12 hours of shaking at37° C., 1.5 mls of the culture was used in the Qiaspin miniprep kit toisolate plasmid DNA. The restriction enzyme EcoR1 was used to confirmthat the plasmid contained a piece of DNA that was approximately 1200bp.

[0492] Sequencing with a T7 primer was performed at the San Diego StateUniversity DNA Core Facility (San Diego, Calif.). The resultant ratEdg-3 sequence are presented as SEQ ID NO.: 7. Rat Edg-3 Sequence (SEQID NO.:7) 0001 ATGGCAACCA CGCACCCGCA CGGGCACCCG CCAGTCTTGG GGAATGATACTCTCCGGGAA 0061 CATTATGATT ACGTGGGGAA GCTGGCAGGC AGGCTGCGGG ATCCCCCTGAGGGTAGCACC 0121 CTCATCACCA CCATCCTCTT CTTGGTCACC TGTAGCTTCA TCGTCTTGGAGAACCTGATG 0181 GTTTTGATTG CCATCTGGAA AAACAATAAA TTTCATAACC GCATGTACTTTTTCATCGGC 0241 AACTTGGCTC TCTGCGACCT GCTGGCCGGC ATAGCCTACA AGGTCAACATTCTGATGTCC 0301 GGTAGGAAGA CGTTCAGCCT GTCTCCAACA GTGTGGTTCC TCAGGGAGGGCAGTATGTTC 0361 GTAGCCCTGG GCGCATCCAC ATGCAGCTTA TTGGCCATTG CCATTGAGCGGCACCTGACC 0421 ATGATCAAGA TGAGGCCGTA CGACGCCAAC AAGAAGCACC GCGTGTTCCTTCTGATTGGG 0481 ATGTGCTGGC TAATTGCCTT CTCGCTGGGT GCCCTGCCCA TCCTGGGCTGGAACTGCCTG 0541 GAGAACTTTC CCGACTGCTC TACCATCTTG CCCCTCTACT CCAAGAAATACATTCCCTTT 0601 CTCATCAGCA TCTTCACAGC CATTCTGGTG ACCATCGTCA TCTTGTACGCGCGCATCTAC 0661 TTCCTGGTCA AGTCCAGCAG CCGCAGGGTG GCCAACCACA ACTCCGAGAGATCCATGGCC 0721 CTTCTGCGGA CCGTAGTGAT CGTGGTGAGC GTGTTCATCG CCTGTTGGTCCCCCCTTTTC 0781 ATCCTCTTCC TCATCGATGT GGCCTGCAGG GCGAAGGAGT GCTCCATCCTCTTCAAGAGT 0841 CAGTGGTTCA TCATGCTGGC TGTCCTCAAC TCGGCCATGA ACCCTGTCATCTACACGCTG 0901 GCCAGCAAAG AGATGCGGCG TGCTTTCTTC CGGTTGGTGT GCGGCTGTCTGGTCAAGGGC 0961 AAGGGGACCC AGGCCTCCCC GATGCAGCCT GCTCTTGACC CGAGCAGAAGTAAATCAAGC 1021 TCCAGTAACA ACAGCAGCAG CCACTCTCCA AAGGTCAAGG AAGACCTGCCCCATGTGGCT 1081 ACCTCTTCCT GCGTCACTGA CAAAACGAGG TCGCTTCAGA ATGGGGTCCTCTGCAAGTGA 1141 CGGTCT

[0493] Molecular Genetic Approaches to Sphingolipid-Based CardiovascularTherapy

[0494] In addition to traditional approaches to therapeutic agents,approaches based on molecular genetics and recombinant DNA technologyare used to produce agents for sphingolipid-based cardiovasculartherapy.

[0495] Dominant Negative Mutant Proteins

[0496] Dominant negative mutant proteins of enzymes that catalyzereactions leading to the production of undesirable, toxic and/orcardiotoxic sphingolipids, or of sphingolipid receptors, are used asagents for sphingolipid-based cardiovascular therapy. Such proteins aredelivered to cardiac or other tissues to decrease sphingolipidproduction and/or to minimize the cardiotoxic effects of circulatingsphingolipids. Enzymes of particular interest include ceramidase,sphingomyelinase and SPH kinase. Receptors of particular interestinclude members of the EDG family of receptors, especially thosepresently known to bind S-1-P, i.e., EDG-1, EDG-3 and EDG-5.

[0497] Dominant negative mutants are prepared in a variety of ways. Ingeneral, dominant negative mutants of proteins retain their ability tointeract with other molecules but have lost some other function presentin the wildtype protein. For example, a dominant negative mutant of amultimeric enzyme involved in sphingolipid metabolism would be one thatretains the ability to form multimers but has a catalytic domain thathas been inactivated by deletion or mutation of amino acid residues inthe catalytic domain. Such deletions and mutations are created bysite-directed mutagenesis, by random mutagenesis, or by any othersuitable procedure.

[0498] One non-limiting example of a dominant negative mutant that maybe used in the invention of the disclosure is a dominant negative mutantof human SPH kinase. This mutation (Gly82Asp), which was created bysite-directed mutagenesis of the presumed catalytic domain of theenzyme, is stated to block the activation of endogenous SPH kinase byTNF-alpha and IL-1-beta (Pitson et al., J. Biol. Chem. 275:33945-33950,2000).

[0499] Antisense

[0500] Antisense oligonucleotides against mRNAs of key sphingolipidproduction enzymes (e.g., sphingomyelinase, ceramidase, sphingosinekinase) are delivered to cardiac and/or other tissues to inhibit orcompletely block the production and/or harmful effects of undesirable,toxic and/or cardiotoxic sphingolipids. Antisense oligonucleotides aredesigned to decrease the expression of key sphingolipid binding proteinsor receptors such as EDG and SCaMPER are delivered to cardiac and/orother tissues to minimize the cardiotoxic effects of circulatingsphingolipids. Ribozymes that degrade such mRNAs are also used.Additionally or alternatively, antisense expression constructs, whichtranscribe RNA molecules antisense in vivo, may be introduced into ananimal, which may be a human, in need thereof.

[0501] Gene Therapy

[0502] Various forms of gene therapy are used to carry outsphingolipid-based cardiovascular therapy according to the invention.The therapeutic nucleic acid molecule (“gene therapy construct”) that isintroduced into a patient in need thereof generally comprises nucleicacids having the following genetic elements: (i) an “open reading frame(ORF),” i.e., a protein-encoding nucleotide sequence that is sought tobe expressed, and (ii) a “gene therapy vector,” which provides thegenetic sequences needed for expression of an ORF introduced thereinto,replication of the vector and/or recombination with other nucleic acids.A gene or protein is said to be “expressed” when it is actively creatingmRNA molecules having the nucleotide sequence of the ORF of a gene(transcription); and, using these mRNA molecules as blueprints,producing proteins having specific amino acid sequences (translation).It is understood in the art that transcription and translation need notoccur contemporaneously. It is also understood in the art that a proteinis one type of gene product, but other gene products that may beexpressed by gene therapy constructs may be nucleic acids, e.g.,antisense transcripts, or structural or enzymatic RNA molecules.

[0503] Expression of proteins that influence sphingolipid concentrationand/or activity via the administration of gene therapy constructscomprising an ORF that encodes a protein of interest is one type of genetherapy. Such proteins may be expressed at a level that is approximatelyequal to what is normally found in healthy individuals, “over-expressed”(i.e., expressed at a level that is greater than the amount that isnormally found in healthy individuals), or constitutively expressed(i.e., expressed at a constant level).

[0504] Another type of gene therapy, in which a genetic deficiency (lossof function of a protein due to mutation) is compensated for by virtueof the in vivo expression of a wildtype protein from a gene therapyconstruct, is generally called “replacement therapy.” This type of genetherapy may be used to treat patients having genetic deficiencies thatreduce the amount, activity, or distribution in enzymes involved insphingolipid metabolism, or in sphingolipid receptors.

[0505] In another type of gene therapy, proteins that degradeundesirable sphingolipids, or stimulators thereof, are overexpressed forthe immediate and/or long-term treatment of cardiac disorders. In orderto achieve gene therapy of this type, gene therapy constructs aredesigned, by way of non-limiting example, to encode and overexpress anenzyme that degrades an undesirable sphingolipid or metabolite thereof.Such enzymes include, e.g., S-1-P lyase, sphingomyelin synthase, SMdeacylase, ceramide synthase, glucosylceramide synthase, ceramidekinase, and S-1-P phosphatase. An indirect way of realize this type ofgene therapy involves the administration of gene therapy constructs thatencode proteins that are inhibitors or activators of enzymes involved insphingolipid metabolism or sphingolipid receptors (see Examples 7 to10).

[0506] In another type of gene therapy, proteins that bind undesirablesphingolipids are overexpressed for the immediate and/or long-termtreatment of cardiac disorders. In order to achieve gene therapy of thistype, gene therapy constructs are designed that encode, by way ofnon-limiting example, antibodies or antibody derivatives directed to anundesirable sphingolipid (Example 6); naturally occurring proteins, suchas lysenin, that bind an undesirable sphingolipid; or polypeptidederivatives of receptors and enzymes that bind undesirable sphingolipids(Example 15).

[0507] Another type of gene therapy involves the administration of genetherapy constructs that express a RNA having a nucleotide sequence thatis the antisense (hybridizing) of all or a portion of an mRNA thatencodes a sphingolipid enzyme that produces, or a receptor thatfacilitates the cellular uptake of, an undesirable sphingolipid. Bindingof the antisense transcript reduces or prevents the expression of themRNA encoding the enzyme or receptor. Such enzymes include but are notlimited to SMase, SPH kinase, ceramidase, cerebrosidase, desaturase,ceramide synthase, ceramide-1-phosphatase, serine palmitoyl transferase,and NADPH-dependent reductase. Such receptors include but are notlimited to members of the EDG family of receptors (Example 15).

[0508] Another type of gene therapy involves the use of gene therapyconstructs that encode and express dominant negative mutants of keysphingolipid production enzymes (e.g., SPH kinase, sphingomyelinase,etc.) or sphingolipid receptors (e.g., the EDG family of receptors).These expression constructs are delivered to cardiac or other tissues inorder to decrease sphingolipid production and/or to minimize the effectsof circulating undesirable, toxic and/or cardiotoxic sphingolipids.

[0509] Gene Therapy Vectors

[0510] Gene therapy constructs for the in vivo production of antisensetranscripts and dominant negative mutants, and for gene therapy, may becontained in any suitable expression vector known in the art, such as aplasmid, cosmid, or viral vector. Viral vectors such as retroviralvectors, adenovirus vectors, herpes simplex virus vectors, vacciniavirus and the like are particularly useful for the administration ofthese expression constructs. The choice of vector and route ofadministering the vector will depend, for example, on the particulartarget cells, tissues and animal (including a human) that are targetedfor drug delivery, and can be determined by those skilled in the art.

[0511] Non-limiting examples of vectors for gene therapy include, ingeneral, those that are nonviral (Li et al., Gene Ther. 7:31-34, 2000;episomal (Van Craenenbroeck et al., Eur. J. Biochem. 267:5665-5678,2000); viral (Walther et al., Drugs 60:249-271, 2000), including, inparticular, those derived from retroviruses (Kurian et al., Mol. Pathol.53:173-176, 2000; Takeuchi et al., Adv. Exp. Med. Biol. 465:23-35, 2000)and other RNA viruses (Hewson, Mol Med. Today 6:28-35, 2000).Non-limiting examples of particular gene therapy vectors include thosederived from adenovirus (see Danthinne et al., Gene Ther. 7:1707-1704,2000); AAV, adeno-associated virus (Athanasopoulus et al., Int. J. Mol.Med. 6:363-375, 2000; Tal, J. Biomed. Sci. 7:279-291, 2000; Monahan etal., Gene Ther. 7:24-30, 2000); HSV-1, herpes simplex virus(Sean-Esteves et al., Mol. Ther. 2:9-15, 2000; Latchman, Mol. Med. Today6:28-35, 2000); and lentiviral vectors (Vigna et al., J. Gene Med.2:308-316, 2000; Buchschacher et al., Blood 95:2499-2504, 2000; Trono,Gene Ther. 7:20-23, 2000).

[0512] Gene therapy vectors and constructs may be designed to betargeted to specific cells or tissues (Hallenbeck et al., Adv. Exp. Med.Biol. 465:37-46, 2000). Gene therapy vectors designed to be targeted tomyocardial cells and tissues, and strategies for the use thereof, arepreferably used in certain modalities of gene therapy. See U.S. Pat. No.6,121,246; Allen, Ann. Thorac. Surg. 68:1924-1928, 1999; Ponder, TrendsCardiovasc. Med. 9:158-162, 1999; Yla-Herttuala et al., Lancet355:213-222, 2000; Duckers et al., Med. Clin. North Am. 84:199-213,2000; Sinnaeve et al., Cardiovasc. Res. 44:498-506, 1999; Hiltunen etal., Vasc. Med. 5:41-48, 2000; Hajjar et al., Circ. Res. 86:616-621,2000; Stephan et al., Ann. Endocrinol. (Paris) 61:85-90, 2000; O'Brienet al., Mayo Clin. Proc. 75:831-834, 2000; Dedieu et al., Curr. Cardiol.Rep. 2:39-47, 2000.

Example 19

[0513] Combination Therapies

[0514] The therapeutic compositions and methods of the invention may beused in combination with each other and/or with other agents forcardiovascular therapy, i.e., non-sphingolipid-based therapeutic agents.In such instances, pharmaceutical compositions, medical devices,emergency kits and the like contain two or more of the therapeuticagents of the invention; or at least one of the therapeutic agents ofthe invention and at least one non-sphingolipid-based therapeutic agent;or two or more of the therapeutic agents of the invention and two ormore other therapeutic agents. That is, compositions, devices, methodsand the like that are used in combination therapies may be described asthose having or using at least one member from two or more of a, b andc, wherein “a” is a first therapeutic agent of the invention; “b” is asecond (i.e., other than “a”) therapeutic agent of the invention; and“c” is a non-sphingolipid-based therapeutic agent.

[0515] In general, any therapeutic agent may be combined with thetherapeutic agents of the invention so long as neither agent has anegative impact on the activity of the other agent. Therapeutic agentsthat may be combined with the therapeutic agents of the inventionincludes presently known agents for cardiovascular therapy, as well asagents that are discovered or created subsequent to the filing of thepresent application.

[0516] Presently known agents for cardiovascular therapy include but arenot limited to alpha and beta adrenergic blocking drugs; parasympatheticdrugs; calcium channel blockers; drugs that affect the renin-angiotensinsystem; diuretic therapy; magnesium, potassium and calcium; digitalisand other inotropic agents; organic nitrates and nitroprusside;antiadrenergic drugs with central action; ganglionic blockers and neurondepletors; nonspecific antihypertensive vasodilators; antiarrhythmicdrugs; antiplatelet and other antithrombotic drugs; thrombolytic agents;lipid-lowering drugs; selective dopamine receptor agonists; andprostacyclin. See Cardiovascular Pharmacotherapeutics: CompanionHandbook, Wm. H. Frishman and Edmund. H Sonnenblick, McGraw Hill, N.Y.,1998.

[0517] As is noted in the preceding Examples, certain therapeutic agentsof the invention are preferably used in combination with a secondtherapeutic agent designed to ameriolate potential undesiredside-effects that may occur as a result of treatment with the firsttherapeutic agent.

[0518] By way of non-limiting example, inhibition of SPH kinase willresult in decreased production of the harmful sphingolipid S-1-P, butmay lead to an accumulation of SPH, which is also an undesirablesphingolipid, albeit generally less harmful than S-1-P. In order toavoid or mitigate this effect should it occur, additional agents areadministered that lower the concentration of available SPH. Such agentsinclude but are not limited to ones that (i) stimulate or are enzymeshaving SPH as a substrate, with the proviso that such enzymes should notbe ones that produce S-1-P; (ii) inhibit enzymes having SPH as aproduct; (iii) are SPH receptor derivatives, or antibodies to SPH, thatbind molecules of SPH, thus sequestering them from locations in the bodywhere they exert their toxic effects; (iii) inhibit the action ofinflammatory cytokines and chemokines; (iv) are antisense molecules, orgenetic constructs that express antisense transcripts, that act toreduce the expression of a protein that increases concentrations of SPH,e.g., an enzyme that catalyzes reactions that produce SPH; (v) adominant negative derivative of a protein that increases concentrationsof SPH, e.g., an enzyme that catalyzes reactions that produce SPH; and(vi) a gene therapy construct that encodes and expresses a protein thatleads to decreased function and/or concentration of SPH including, byway of non-limiting example, a protein that is characterized as being ofthe type encompassed by the classes defined above in (i), (ii), (iii)and/or (v).

[0519] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themethods, procedures, treatments, devices, and compositions describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Upon reading this specification, changes therein and otheruses will occur to those skilled in the art, each of which isencompassed within the spirit of the invention as defined by theattached claims.

[0520] All patents and publications referred to above are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0521] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, or limitation orlimitations, which is not specifically disclosed herein.

[0522] Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims. Other embodiments arewithin the following claims.

1. A nucleic acid having the nucleotide sequence SEQ ID NO:
 3. 2. Apolypeptide encoded by the nucleic acid of claim
 1. 3. A nucleic acidhaving the nucleotide sequence SEQ ID NO:
 4. 4. A polypeptide encoded bythe nucleic acid of claim
 3. 5. A nucleic acid having the nucleotidesequence SEQ ID NO:
 7. 6. A polypeptide encoded by the nucleic acid ofclaim
 5. 7. An expression construct comprising a nucleic acid having asequence selected from the group consisting of SEQ ID NOS: 3, 4, and 7.8. A host cell comprising the expression construct of claim
 7. 9. Amethod of producing a polypeptide, comprising culturing the host cell ofclaim
 8. 10. An expression construct comprising a portion of a nucleicacid having a portion of sequence selected from the group consisting ofSEQ ID NO: 3 wherein said portion of said sequence lacks nucleotidesequences that encode a transmembrane domain of a polypeptide encodedthereby.
 11. An expression construct comprising a portion of a nucleicacid having a portion of sequence selected from the group consisting ofSEQ ID NO: 4 wherein said portion of said sequence lacks nucleotidesequences that encode a transmembrane domain of a polypeptide encodedthereby.
 12. An expression construct comprising a portion of a nucleicacid having a portion of sequence selected from the group consisting ofSEQ ID NO: 7 wherein said portion of said sequence lacks nucleotidesequences that encode a transmembrane domain of a polypeptide encodedthereby.
 11. A host cell comprising the expression vector of claim 10.12. A host cell comprising the expression vector of claim
 11. 13. A hostcell comprising the expression vector of claim
 12. 14. A method ofproducing a soluble receptor fragment, comprising culturing the hostcell of claim
 11. 15. A method of producing a soluble receptor fragment,comprising culturing the host cell of claim
 12. 16. A method ofproducing a soluble receptor fragment, comprising culturing the hostcell of claim
 13. 17. A soluble receptor fragment, derived from areceptor selected from the group consisting of Edg-1, Edg-3, Edg-5,Edg-6, Edg-8, the Mil receptor, AXOR29, NRG1, SCaMPER and homologs andisoforms thereof.
 18. A method of screening for an agent for treating orpreventing cardiovascular or cerebrovascular disease, comprisingscreening a library of compounds for agents that bind a receptor forsphingolipid or a sphingolipid metabolite.
 19. The method of claim 18wherein said receptor is selected from the group consisting of Edg-1,Edg-3, Edg-5, Edg-6, Edg-8, the Mil receptor, AXOR29, NRG1, SCaMPER andhomologs and isoforms thereof.
 20. The method of claim 18 wherein saidreceptor is encoded by SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7.